Modified ligand-dependent ion channel, and method for using the same

Abstract

To provide a material and a method for adjusting ligand-dependent ion channel (LGIC) activity which can be used for adjusting the excitability of cells in a mammal.SOLUTION: Provided is a modified LGIC including at least one LGIC subunit having a modified ligand binding domain (LBD) and / or a modified ion pore domain (IPD), for example. Also provided are an exogenous LGIC ligand that can bind to the modified LGIC to activate it, and a method for adjusting ion transport crossing a membrane of a mammalian cell, a method for adjusting the excitability of cells in a mammal, and a method for treating a mammal having channel disorder.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 62 / 584,428, filed November 10, 2017, and U.S. Patent Application No. 62 / 729,716, filed September 11, 2018. The disclosures of these prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.

[0002] 1.Technical Field The present specification relates to materials and methods for modulating ligand-gated ion channel (LGIC) activity. For example, the present specification provides modified LGICs including at least one LGIC subunit having a modified ligand binding domain (LBD) and / or a modified ion pore domain (IPD). Also provided are exogenous LGIC ligands capable of binding to and activating the modified LGIC. In certain cases, the modified LGICs and exogenous ligands can be used to treat mammals with channelopathies (e.g., nerve channelopathies or muscle channelopathies). In certain cases, the modified LGICs and exogenous LGIC ligands can be used to modulate (e.g., activate or inhibit) ion transport across membranes of mammalian cells. In certain cases, the modified LGICs and exogenous LGIC ligands can be used to modulate (e.g., increase or decrease) cellular excitability in mammals. [Background technology]

[0003] 2. Background information Ion channels mediate ion flux within cells, which profoundly influences their biological function. A prominent example of this is in neurons, where ion channels control electrical signaling within and / or between neurons to affect physiology, sensation, behavior, mood, and cognition.

[0004] Different LGICs have distinct ligand-binding and ion-conducting properties (Hille 2001 Ion Channels of Excitable Membranes. pp. 814. Sunderland, MA: Sinauer Associates; Kandel et al. 2000 Principles of Neural Science. USA: McGraw-Hill Co. pp. 1414). For example, nicotinic acetylcholine receptors (nAChRs) bind the endogenous ligand acetylcholine (ACh), which activates a cation conductance, typically depolarizing the cell and thereby increasing cell excitability. In contrast, glycine receptors (GlyRs) bind the endogenous ligand glycine, which activates a chloride anion conductance, typically decreasing cell excitability by hyperpolarizing and / or electrical shunting across cell membrane resistance. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Hille 2001 Ion Channels of Excitable Membranes. pp. 814. Sunderland, MA: Sinauer Associates. [Non-patent document 2] Kandel et al 2000 Principles of Neural Science. USA: McGraw-Hill Co. 1414 pp Summary of the Invention

[0006] overview Levels of endogenous LGIC ligands (eg, agonists) such as ACh are not easily controlled. The present specification provides materials and methods for modulating LGIC activity (e.g., increasing the sensitivity of LGIC to exogenous ligands and / or decreasing the sensitivity to endogenous ligands such as ACh). For example, the present specification provides modified LGICs comprising at least one modified LGIC subunit having an LBD and an IPD and at least one modified amino acid (e.g., an amino acid substitution). Exogenous LGIC ligands that can bind to and modulate (e.g., activate) the modified LGIC are also provided. In certain cases, the modified LGICs and exogenous ligands can be used to treat mammals with channelopathies (e.g., nerve or muscle channelopathies). In certain cases, the modified LGICs and exogenous LGIC ligands can be used to modulate (e.g., activate or inhibit) ion transport across membranes of mammalian cells. In certain cases, the modified LGICs and exogenous LGIC ligands can be used to modulate (e.g., increase or decrease) cellular excitability in mammals.

[0007] The ability to control LGIC activity offers unique and unrealized opportunities for controlling intracellular ion transport. For example, modified LGICs with increased sensitivity to one or more exogenous LGIC ligands can be used to provide temporal and spatial control of ion transport and / or cell excitability based on the delivery of exogenous LGIC ligands. For example, modified LGICs with reduced sensitivity to endogenous LGIC ligands prevent undesired activation of the modified LGIC and enable selective control of the modified LGIC by exogenous ligands. Furthermore, exogenous LGIC ligands with enhanced potency for the modified LGIC improve the selectivity of targeting the modified LGIC to endogenous ion channels. Thus, the modified LGICs and exogenous LGIC ligands provided herein are useful for achieving therapeutic effects while reducing side effects from small molecules targeting unintended targets.

[0008] As described herein, one or more mutations in the modified LGIC can enhance the potency of exogenous LGIC ligands. Mutation of the α7 LBD of α7-GlyR at residue L131 (e.g., replacing Leu with Gly or Ala) reduced the potency of ACh against α7-GlyR (-6.4-fold) while increasing the potency of varenicline (16-fold) and tropisetron (3.6-fold). Mutation of the α7 LBD of α7-GlyR at residues G175 (e.g., G175K) or P216 (e.g., P216I) enhanced the potency of ACh, nicotine, tropisetron, varenicline, and other quinuclidine and tropane agonists. Combining a mutation at residue G175K with a mutation that reduces the potency of the endogenous agonist ACh (e.g., Y115F) generated α7-GlyR Y115F G175K, which increased potency against tropisetron (5.5-fold) and decreased potency against ACh (-8-fold). Furthermore, mutations in the α7 LBD at residues 77 (e.g., Trp to Phe or Tyr), 79 (e.g., Gln to Gly, Ala, or Ser), 131 (e.g., Leu to Gly or Ala), and / or 141 (e.g., Leu to Phe or Pro) in these chimeric channels combined with potency-enhancing mutations at residues G175 (e.g., G175K) or P216 (e.g., P216I) increased potency against different ligands and / or decreased ACh potency. For example, a chimeric α7-GlyR LGIC having an α7 nAChR LBD (α7 LBD) with mutations at residue 79 (e.g., Gln to Gly), residue 115 (e.g., Tyr to Phe), and residue 175 (e.g., Gly to Lys) has a greater than 100-fold increased sensitivity to the exogenous tropane LGIC ligand Compound 723 (Tropane) and a decreased ACh sensitivity (-15-fold) relative to the unmodified chimeric α7-GlyR LGIC.Furthermore, modified LGICs containing at least one chimeric LGIC subunit with an α7 nAChR LBD (α7 LBD) with a mutation at residue 79 (e.g., substituting Gln for Ala, Gly, or Ser) and a GlyR IPD with a mutation at residue 298 (e.g., substituting Ala for Gly) have nearly 20-fold increased sensitivity to exogenous LGIC ligands such as quinuclidine or tropane. Further mutations at residues 27 (e.g., substituting Arg for Asp) and 41 (e.g., substituting Glu for Arg) of the α7 LBD reduced the association of the modified chimeric LGIC with unmodified ion channels. Additional mutations at residues 115 (e.g., Tyr for Phe), 139 (e.g., Gln for Gly or Leu), 210 (e.g., Tyr for Phe), 217 (e.g., Tyr for Phe), and / or 219 (e.g., Asp for Ala) of the α7 LBD reduced the sensitivity of the chimeric LGIC to the endogenous ligand ACh. These chimeric LGICs enable highly selective control of cellular functions in mammalian cells while minimizing cross-reactivity with endogenous mammalian signaling systems.

[0009] In general, one aspect of the present disclosure features a modified LGIC having at least one modified LGIC subunit including an LBD with an amino acid modification and an IPD, wherein an exogenous LGIC ligand activates the modified LGIC. The modified LGIC may be a chimeric LGIC having an LBD from a first LGIC and an IPD from a second LGIC. The LBD may be an alpha7 nicotinic acetylcholine receptor (α7-nAChR) LBD. The modified LGIC of claim 3, wherein at least one modified amino acid in the α7-nAChR LBD comprises an amino acid substitution at an amino acid residue selected from the group consisting of residues 77, 79, 131, 139, 141, 175, and 216 of the α7-nAChR LBD. The amino acid substitution may be at residue 79 of the α7 LBD, and the amino acid substitution may be Q79A, Q79G, or Q79S. For example, the amino acid substitution at residue 79 of the α7 LBD may be Q79G. The IPD can be a serotonin 3 receptor (5HT3) IPD, a glycine receptor (GlyR) IPD, a gamma-aminobutyric acid (GABA) receptor IPD, or an α7-nAChR IPD. The IPD can be a GlyR IPD, which can include an amino acid substitution (e.g., an A298G substitution) at residue 298 of the chimeric LGIC. The IPD can be a GABA IPD, which can include an amino acid substitution (e.g., a W298A substitution) at residue 298 of the modified LGIC. The modified LGIC can be a chimeric LGIC comprising an α7 LBD with a Q79G amino acid substitution and a GlyR IPD with an A298G amino acid substitution. The exogenous LGIC ligand can be a synthetic exogenous LGIC ligand selected from the group consisting of quinuclidine, tropane, 9-azabicyclo[3.3.1]nonane, 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, and 1,4-diazabicyclo[3.2.2]nonane. When the synthetic exogenous LGIC ligand is a tropane, the tropane can be tropisetron, pseudotropisetron, nortropisetron, Compound 723, Compound 725, Compound 737, or Compound 745.When the synthetic exogenous LGIC ligand is quinuclidine, the quinuclidine can be PNU-282987, PHA-543613, Compound 0456, Compound 0434, Compound 0436, Compound 0354, Compound 0353, Compound 0295, Compound 0296, Compound 0536, Compound 0676, or Compound 702. When the synthetic exogenous LGIC ligand is 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, the ligand can be Compound 765 or Compound 770. When the synthetic exogenous LGIC ligand is 1,4-diazabicyclo[3.2.2]nonane, the ligand can be Compound 773 or Compound 774. In certain cases, the LBD may be an α7 LBD, and the α7 LBD may also contain at least one modified amino acid that confers selective binding to another α7 LBD having at least one modified amino acid over binding to an unmodified LGIC. The unmodified LGIC may be an endogenous LGIC (e.g., an endogenous α7-nAChR). The at least one modified amino acid in the α7 LBD that results in reduced binding to an unmodified LGIC may include an amino acid substitution at residue 27 (e.g., an R27D substitution) and / or residue 41 (e.g., an E41R substitution). In some cases, the IPD may be a 5HT3 IPD, and the 5HT3 IPD may contain at least one modified amino acid that confers increased ionic conductivity (conductance) to the modified LGIC. The at least one modified amino acid in the 5HT3 IPD that confers increased ionic conductivity to the modified LGIC can include an amino acid substitution at amino acid residues 425 (e.g., an R425Q substitution), 429 (e.g., an R429D substitution), and / or 433 (e.g., an R433A substitution).

[0010] In another aspect, the present disclosure features a modified LGIC having at least one modified LGIC subunit comprising an LBD and an IPD with at least one modified amino acid, wherein the at least one modified amino acid in the LBD reduces binding to an endogenous LGIC ligand. The modified LGIC can be a chimeric LGIC having an LBD from a first LGIC and an IPD from a second LGIC. The endogenous LGIC ligand can be ACh. The modified LGIC can have an EC50 of greater than 20 μM for ACh. The at least one modified amino acid can include an amino acid substitution at residues 115, 139, 210, 217, and / or 219. When the at least one modified amino acid includes an amino acid substitution at residue 115, the amino acid substitution can be a Y115F substitution. When the at least one modified amino acid includes an amino acid substitution at residue 139, the amino acid substitution can be a Q139G or Q139L substitution. When the at least one modified amino acid includes an amino acid substitution at residue 210, the amino acid substitution can be a Y210F substitution. When at least one modified amino acid comprises an amino acid substitution at residue 217, the amino acid substitution can be a Y217F substitution. When at least one modified amino acid comprises an amino acid substitution at residue 219, the amino acid substitution can be a D219A substitution. In certain cases, a modified LGIC can comprise a modified α7-nAChR LBD having an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution. A modified LGIC (e.g., a modified LGIC comprising a modified α7-nAChR LBD having an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution) can also comprise an endoplasmic reticulum transport sequence. The endoplasmic reticulum transport sequence can comprise the amino acid sequence: FCYENEV (SEQ ID NO: 16). For example, a modified LGIC comprising a modified α7-nAChR LBD having an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution, and also comprising an endoplasmic reticulum transport sequence, can comprise the amino acid sequence set forth in SEQ ID NO: 13. A modified LGIC (e.g., a modified LGIC comprising a modified α7-nAChR LBD having an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution) can comprise a signal sequence (e.g., a CHRNB4 signal sequence).The CHRNB4 signal sequence has the amino acid sequence: TIFF2025131833000001.tif4128. For example, a modified LGIC comprising a modified α7-nAChR LBD having an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution, and also comprising a CHRNB4 signal sequence, may comprise the amino acid sequence set forth in SEQ ID NO: 14. A modified LGIC (e.g., a modified LGIC comprising a modified α7-nAChR LBD having an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution) may also comprise a somatic targeting sequence (e.g., a KCNB1 somatic targeting sequence). The KCNB1 somatic targeting sequence has the amino acid sequence: TIFF2025131833000002.tif12158. For example, a modified LGIC comprising a modified α7-nAChR LBD having an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution, and also comprising a KCNB1 somatic targeting sequence, can comprise the amino acid sequence set forth in SEQ ID NO:15.

[0011] In another aspect, this document features a ligand with increased potency for a modified ligand-gated ion channel (LGIC), wherein the ligand has a structure represented by Formula I: TIFF2025131833000003.tif32128 (wherein each of X1, X2, and X3 can independently be CH, CH2, O, NH, or NMe; each n can independently be 0 or 1; Y is O or S; A is an aromatic substituent; and R is H or pyridinylmethylene.) The aromatic substituent can be 1H-indole, 4-(trifluoromethyl)benzene, 2,5-dimethoxybenzene, 4-chloroaniline, aniline, 5-(trifluoromethyl)pyridin-2-yl, 6-(trifluoromethyl)nicotinic, or 4-chlorobenzene.

[0012] In certain cases, the LGIC ligand can be a quinuclidine, represented by formula II: TIFF2025131833000004.tif18128 (wherein X3 is O, NH, or CH2, Y is O or S, A is an aromatic substituent, and R is H or pyridinylmethylene) The aromatic substituent may be 1H-indole, 4-(trifluoromethyl)benzene, 4-chlorobenzene, 2,5-dimethoxybenzene, 4-(trifluoromethyl)benzene, 4-chloroaniline, aniline, 5-(trifluoromethyl)pyridin-2-yl, 6-(trifluoromethyl)nicotine, 3-chloro-4-fluorobenzene, or 1H-indole. The quinuclidine may be PNU-282987, PHA-543613, Compound 0456, Compound 0434, Compound 0436, Compound 0354, Compound 0353, Compound 0295, Compound 0296, Compound 0536, Compound 0676, or Compound 702.

[0013] In certain cases, the LGIC ligand may be a tropane and has formula III: TIFF2025131833000005.tif29128 (wherein X2 is NH or NMe, X3 is O, NH, or CH2, Y is O or S, and A is an aromatic substituent) The aromatic substituent can be 1H-indole, 7-methoxy-1H-indole, 7-methyl-1H-indole, 5-chloro-1H-indole, or 1H-indazole. The tropane can be tropisetron, pseudotropisetron, nortropisetron, Compound 723, Compound 725, Compound 737, or Compound 745.

[0014] In certain cases, the LGIC ligand can be 9-azabicyclo[3.3.1]nonane and has the formula IV: TIFF2025131833000006.tif27128 (wherein X1 can be CH, X2 can be NH or NMe, X3 can be O, NH, or CH, Y can be O or S, and A can be an aromatic substituent) The aromatic substituent can be 4-chloro-benzene. 9-Azabicyclo[3.3.1]nonane can be compound 0536.

[0015] In another aspect, provided herein is a ligand having increased potency for modified LGICs, wherein the ligand can be a 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine and has formula V: The present invention features a ligand that can have the structure shown in TIFF2025131833000007.tif14128 (wherein R1 can be H, phenyl, 2-toluyl, 3-pyridyl, 4-pyridyl, trifluoromethyl, methoxy, N,N-dimethylamino, N,N-diethylamino, imidazole, pyrrole, pyrazole, triazole, or isoxazol-3-amine, and R2 can be H, methyl, or phenyl). The 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine can be varenicline, Compound 0765, Compound 0770, Compound 0780, Compound 0782, Compound 0785, Compound 0788, Compound 0782, Compound 0789, Compound 0791, Compound 0793, Compound 0794, Compound 0795, Compound 0798, Compound 0799, Compound 0800, Compound 0801, Compound 0802, Compound 0803, Compound 0804, Compound 0805, Compound 0807, Compound 0808, Compound 0812, Compound 0813, Compound 815, and Compound 817.

[0016] In another aspect, provided herein is a ligand having increased potency for modified LGICs, the ligand being 2-(pyridin-3-yl)-1,5,6,7,8,9-hexahydro-5,9-methanoimidazo[4',5':4,5]benzo[1,2-d]azepine (compound 0786): The ligand may be characterized as TIFF2025131833000008.tif12128.

[0017] In another aspect, provided herein is a ligand having increased potency against modified LGICs, which can be 7,8,9,10-tetrahydro-1H-6,10-methanoazepino[4,5-g]quinoxalin-2(6H)-one and has formula VI: The present invention features a ligand that can have the structure shown in TIFF2025131833000009.tif16128, where R can be H or CH, R can be H, CH, or an aromatic substituent, and R can be O or S. The 7,8,9,10-tetrahydro-1H-6,10-methanoazepino[4,5-g]quinoxalin-2(6H)-one can be Compound 0783, Compound 0784, Compound 0790, or Compound 0792.

[0018] In another aspect, this document features a ligand with increased potency for modified LGICs, where the ligand can be 1,4-diazabicyclo[3.2.2]nonane and has Formula VII: TIFF2025131833000010.tif25128 (where R can be H, F, or NO2) The 1,4-diazabicyclo[3.2.2]nonane can be 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-dioxide, compound 0773, or compound 0774.

[0019] In another aspect, this document features a method for treating a channelopathy in a mammal. The method includes, or consists essentially of, administering a modified LGIC to a cell in the mammal, wherein an exogenous LGIC ligand selectively binds to the modified LGIC. The modified LGIC has at least one modified LGIC subunit including an LBD and an IPD that include at least one modified amino acid. The exogenous ligand is then administered to the mammal. The channelopathy can be Bartter syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT), congenital hyperinsulinemia, cystic fibrosis, Dravet syndrome, episodic ataxia, erythromelalgia, generalized epilepsy (e.g., with febrile seizures), familial hemiplegic migraine, fibromyalgia, hyperkalemic periodic paralysis, hypokalemic periodic paralysis, Lambert-Eaton myasthenic syndrome, long QT syndrome (e.g., Romano-Ward syndrome), short QT syndrome, malignant hyperthermia, mucolipidosis type IV, myasthenia gravis, myotonia congenita, neuromyelitis optica, neuromyotonia, nonsyndromic hearing loss, congenital paramyotonia, retinitis pigmentosa, Timothy syndrome, tinnitus, seizures, trigeminal neuralgia, and multiple sclerosis.

[0020] In another aspect, the present disclosure features a method for modulating ion transport across a mammalian cell membrane. The method comprises or consists essentially of administering a modified LGIC to a cell, wherein an exogenous LGIC ligand selectively binds to the modified LGIC. The modified LGIC has at least one modified LGIC subunit comprising an LBD and an IPD that include at least one modified amino acid. The exogenous ligand is then administered to the mammal. The modulation can include activating or inhibiting ion transport. The cell can be a neuron, glial cell, muscle cell, stem cell, endocrine cell, or immune cell. The administration of the modified LGIC to the cell can be in vivo or ex vivo. The administration of the modified LGIC to the cell can include administration of a nucleic acid encoding the modified LGIC.

[0021] In another aspect, the present disclosure features a method for modulating the excitability of a cell in a mammal. The method comprises or consists essentially of administering a modified LGIC to a cell derived from the mammal, wherein an exogenous LGIC ligand selectively binds to the modified LGIC. The modified LGIC has at least one modified LGIC subunit comprising an LBD and an IPD that include at least one modified amino acid. The exogenous ligand is then administered to the mammal. The modulation can include increasing or decreasing the excitability of the cell. The cell can be an excitable cell. The cell can be a neuron, glial cell, muscle cell, stem cell, endocrine cell, or immune cell. The administration of the modified LGIC to the cell can be in vivo or ex vivo. The administration of the modified LGIC to the cell can include administration of a nucleic acid encoding the modified LGIC.

[0022] In another aspect, the description features a method for modulating cellular activity in a mammal. The method comprises or consists essentially of administering a modified LGIC to a cell, wherein an exogenous LGIC ligand selectively binds to the modified LGIC. The modified LGIC has at least one modified LGIC subunit comprising an LBD and an IPD that include at least one modified amino acid. The exogenous ligand is then administered to the mammal. The modulation can include increasing or decreasing cellular activity. The activity can be ion transport, passive transport, excitation, inhibition, or exocytosis. The cell can be a neuron, glial cell, muscle cell, stem cell, endocrine cell, or immune cell. The administration of the modified LGIC to the cell can be in vivo or ex vivo. The administration of the modified LGIC to the cell can include administration of a nucleic acid encoding the modified LGIC (e.g., via a viral vector such as an adeno-associated virus, herpes simplex virus, or lentivirus).

[0023] In another aspect, this document features a method for identifying a ligand that selectively binds to a modified LGIC. The method comprises, or consists essentially of, providing one or more candidate ligands to a modified LGIC described herein and detecting binding between the candidate ligands and the modified LGIC, thereby identifying a ligand that selectively binds to the modified LGIC. The modified LGIC can be a homomeric modified LGIC.

[0024] In another aspect, the description features a method for detecting a modified LGIC. The method comprises, or consists essentially of, providing one or more modified LGIC subunits described herein, providing an agent that selectively binds to the modified LGIC, and detecting the modified LGIC by detecting binding between the modified LGIC and the agent that selectively binds to the modified LGIC. The agent that selectively binds to the modified LGIC can be an antibody, a protein (e.g., bungarotoxin), or a small molecule (e.g., a positron emission tomography (PET) ligand). The agent that selectively binds to the modified LGIC can include a detectable label (e.g., a fluorescent label, a radioactive label, or a positron-emitting label).

[0025] In another aspect, the present disclosure features a synthetic nucleic acid construct comprising a nucleic acid sequence having at least 75% sequence identity to the sequence set forth in SEQ ID NO:27. The nucleic acid sequence having at least 75% sequence identity to the sequence set forth in SEQ ID NO:27 can include a nucleic acid sequence capable of encoding an LBD having an amino acid modification. The modified LBD can be an α7-nAChR LBD having amino acid substitution residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, and / or 219 of the α7-nAChR LBD. The nucleic acid sequence having at least 75% sequence identity to the sequence set forth in SEQ ID NO:27 can include a nucleic acid sequence capable of encoding an IPD. The IPD can be a 5HT3 IPD, a GlyR IPD, a GABA receptor IPD, or an α7-nAChR IPD. The IPD can be a 5HT3 IPD.

[0026] In another aspect, this document features a synthetic nucleic acid construct including a nucleic acid sequence having at least 75% sequence identity to the sequence set forth in SEQ ID NO:28.

[0027] A nucleic acid sequence having at least 75% sequence identity to the sequence set forth in SEQ ID NO:28 may include a nucleic acid sequence capable of encoding an LBD having an amino acid modification. The modified LBD may be an α7-nAChR LBD having amino acid substitution residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, and / or 219 of the α7-nAChR LBD. A nucleic acid sequence having at least 75% sequence identity to the sequence set forth in SEQ ID NO:28 may include a nucleic acid sequence capable of encoding an IPD. The IPD may be a 5HT3 IPD, a GlyR IPD, a GABA receptor IPD, or an α7-nAChR IPD. The IPD may be a GlyR IPD having an amino acid modification. The modified GlyR IPD may have an amino acid substitution at amino acid residue 298 of the GlyR IPD.

[0028] In another aspect, the present disclosure features a synthetic nucleic acid construct comprising a nucleic acid sequence having at least 75% sequence identity to the sequence set forth in SEQ ID NO:29. The nucleic acid sequence having at least 75% sequence identity to the sequence set forth in SEQ ID NO:29 can include a nucleic acid sequence capable of encoding an LBD having an amino acid modification. The modified LBD can be an α7-nAChR LBD having amino acid substitution residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, and / or 219 of the α7-nAChR LBD. The nucleic acid sequence having at least 75% sequence identity to the sequence set forth in SEQ ID NO:29 can include a nucleic acid sequence capable of encoding an IPD. The IPD can be a 5HT3 IPD, a GlyR IPD, a GABA receptor IPD, or an α7-nAChR IPD. The IPD can be a GABA IPD having an amino acid modification. The modified GABA IPD can have an amino acid substitution at amino acid residue 298 of the GABA IPD.

[0029] In another aspect, this document features a synthetic nucleic acid construct having the sequence set forth in SEQ ID NO:33.

[0030] In another aspect, this document features a synthetic nucleic acid construct having the sequence set forth in SEQ ID NO:34.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Methods and materials are described herein for use in this disclosure.Other suitable methods and materials known in the art can also be used.Materials, methods, and examples are illustrative only and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will control.

[0032] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0033] [Figure 1A]Figure 1 shows exemplary amino acid sequences of chimeric LGICs. Mutations at amino acid residue 77 (e.g., W77F or W77Y) conferred sensitivity to granisetron and tropisetron. Mutations at amino acid residue 79 (e.g., Q79G) were most effective against several agonists. Mutations at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. Potency was significantly enhanced when LBD mutations were combined with mutations at amino acid residue 298 of the GlyR or GABAC IPD. Potency was also enhanced when α7 nAChR LBD mutations were combined with mutations at amino acid residues G175 and P216. A) Amino acid sequence of the α7-5HT3 chimeric receptor (SEQ ID NO:6) comprising the human α7 nAChR LBD (SEQ ID NO:1) and mouse 5HT3 IPD (SEQ ID NO:3) constructs. [Figure 1B] Figure 1 shows exemplary amino acid sequences of chimeric LGICs. Mutations at amino acid residue 77 (e.g., W77F or W77Y) conferred sensitivity to granisetron and tropisetron. Mutations at amino acid residue 79 (e.g., Q79G) were most effective against several agonists. Mutations at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. Potency was significantly enhanced when LBD mutations were combined with mutations at amino acid residue 298 of the GlyR or GABAC IPD. Potency was also enhanced when α7 nAChR LBD mutations were combined with mutations at amino acid residues G175 and P216. B) Amino acid sequence of the α7-GlyR chimeric receptor (SEQ ID NO: 7), comprising the human α7 nAChR LBD (SEQ ID NO: 2) and human GlyR IPD (SEQ ID NO: 5) constructs. [Figure 1C]Figure 1 shows exemplary amino acid sequences of chimeric LGICs. Mutations at amino acid residue 77 (e.g., W77F or W77Y) conferred sensitivity to granisetron and tropisetron. Mutations at amino acid residue 79 (e.g., Q79G) were most effective against several agonists. Mutations at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. Potency was significantly enhanced when LBD mutations were combined with mutations at amino acid residue 298 of the GlyR or GABAC IPD. Potency was also enhanced when α7 nAChR LBD mutations were combined with mutations at amino acid residues G175 and P216. C) Amino acid sequence of the α7-5HT3 chimeric receptor (SEQ ID NO:8) comprising the human α7 nAChR LBD (SEQ ID NO:1) and human 5HT3 IPD (SEQ ID NO:4) constructs. [Figure 1D] Figure 1 shows exemplary amino acid sequences of chimeric LGICs. Mutations at amino acid residue 77 (e.g., W77F or W77Y) conferred sensitivity to granisetron and tropisetron. Mutations at amino acid residue 79 (e.g., Q79G) were most effective against several agonists. Mutations at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. Potency was significantly enhanced when LBD mutations were combined with mutations at amino acid residue 298 of the GlyR or GABAC IPD. Potency was also enhanced when α7 nAChR LBD mutations were combined with mutations at amino acid residues G175 and P216. D) Amino acid sequence of the α7-GABAc chimeric receptor (SEQ ID NO: 10) comprising the human α7 nAChR LBD (SEQ ID NO: 11) and human GABAC IPD (SEQ ID NO: 9) constructs. [Figure 1E]Figure 1 shows exemplary amino acid sequences of chimeric LGICs. Mutation of amino acid residue 77 (e.g., W77F or W77Y) conferred sensitivity to granisetron and tropisetron. Mutation of amino acid residue 79 (e.g., Q79G) was most effective against some agonists. Mutation of amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. Potency was significantly enhanced when LBD mutations were combined with mutations at amino acid residue 298 of the GlyR or GABAC IPD. Potency was also enhanced when α7 nAChR LBD mutations were combined with mutations at amino acid residues G175 and P216. E) Amino acid sequence of rat nAChR sequence (SEQ ID NO: 12). [Figure 2] Figure 2 shows the EC50 of tropisetron for the α7-5HT3 chimeric LGIC and variants of the chimeric LGIC with LBD mutations at the positions shown in Figure 1. Multiple mutations at Gln79 showed similar or improved potency compared to the unmodified α7-5HT3 channel (arrows). [Figure 3A] Figure 3 shows the relative potency of known nAChR agonists on α7-5HT3 chimeric LGICs. A) Graph of EC50 normalized to unmodified α7-5HT3 chimeric channels (logarithmic scale). *P<0.05 indicates a statistically significant change in potency (ANOVA followed by Dunn's test). [Figure 3B] Figure 3 shows the relative potency of known nAChR agonists against α7-5HT3 chimeric LGIC. B) Chemical structures of known nAChR agonists. [Figure 4]Figure 4 shows the relative potency of known nAChR agonists on α7-GlyR chimeric LGICs. A) Graph of the EC50 for the Q79 LBD mutant normalized to the unmodified α7-GlyR chimeric channel (logarithmic scale). B) Graph of the EC50 for the A298G IPD mutation normalized to the unmodified α7-GlyR chimeric channel (logarithmic scale). C) Graph of the EC50 for α7-GlyRA298G normalized to the unmodified α7-GlyR chimeric channel and compared to the double mutant channel α7Q79G-GlyRA298G (logarithmic scale). *P<0.05, indicating a statistically significant change in potency (ANOVA followed by Dunn's test). [Figure 5A] Figure 5 shows the schematic structures of LGIC agonists with substitution patterns most compatible with potency enhancement for α7Q79G-5HT3 and α7Q79G-GlyRA298G. A) Generalized structure showing properties associated with enhanced potency. [Figure 5B] Figure 5 shows the schematic structures of LGIC agonists with substitution patterns most compatible with potency enhancement for α7Q79G-5HT3 and α7Q79G-GlyRA298G. B) The specific pharmacophores represented in (A) are quinuclidine, tropane, and 9-azabicyclo[3.3.1]nonane core structures. [Figure 5C] Figure 5 shows the schematic structures of LGIC agonists with substitution patterns most compatible with potency enhancement for α7Q79G-5HT3 and α7Q79G-GlyRA298G. C) Exemplary synthetic molecules showing high potency for α7Q79G-GlyRA298G, α7Q79G, Y115F, G175K-GlyR, α7W77F, Q79G, G175K-GlyR. [Figure 6A]Figure 6 shows mutations that reduce the association of the chimeric LCIG α7 nAChR LBD with the unmodified LBD. A) Charge-reversal schematic potential configuration for transfection of two epitope-tagged (HA and V5) constructs encoding α7-5HT3 (top) or two constructs encoding α7-5HT3-HA and α7R21D, E41R-5HT3-V5. Here, association between the two different epitope-tagged subunits would be unfavorable due to the charge-reversal mutations at the subunit interface. [Figure 6B] Figure 6 shows mutations that reduce the association of the chimeric LCIG α7 nAChR LBD with the unmodified LBD. B) Whole-cell recordings in HEK cells expressing V5 epitope-tagged α7R21D, E41R-5HT3 show a robust response to PNU-282987. [Figure 6C] Figure 6 shows mutations that reduce the association of the chimeric LCIG α7 nAChR LBD with the unmodified LBD. C) Association of the α7-5HT3 LGIC with the HA and V5 epitope tags in HEK cells was examined by HA immunoprecipitation (left) or total lysate isolation followed by Western blotting with either anti-HA (top) or anti-V5 antibodies (bottom). In cells coexpressing the channel with the HA and V5 epitopes, anti-HA IP followed by anti-V5 immunoblotting showed coimmunoprecipitation of each type of unmodified channel, but not the charge-reversal mutations in the LBD α7R21D and E41R-5HT3-V5. The MW of α7-5HT3 is approximately 48 kD (arrow). [Figure 7] Figure 7 shows that chimeric LGICs can be regulated using exogenous ligands. Cortical neurons from mouse brains transduced with α7Q79G-GlyRA298G chimeric LGICs via adeno-associated viral (AAV) vectors fire action potentials in response to a 40 pA current injection (PRE), which is potently suppressed by 30 nM tropisetron. After washout of tropisetron (WASH), neuronal firing recovers. [Figure 8A]Figure 8 shows the activity of agonists against chimeric LGICs with the G175K mutation. A) Graph of the EC50 for the Q79G G175K LBD mutant against known agonists (logarithmic scale), normalized to the unmodified α7-GlyR chimeric channel. [Figure 8B] Figure 8 shows the activity of agonists for a chimeric LGIC with a G175K mutation. B) Graph of EC50 for ACh and tropisetron for channels with mutations in an α7-GlyR chimeric LGIC. Mutations resulting in channels with high potency for tropisetron and low potency for the endogenous ligand, acetylcholine (ACh), are optimal (gray shading). Unmod: Unmodified α7-GlyR chimeric LGIC. [Figure 8C] Figure 8 shows the activity of agonists against chimeric LGICs containing the G175K mutation. C) Action potentials of cortical neurons from mouse brains transduced with α7Q79G, Y115F, G175K-GlyR chimeric LGICs. The neurons fire in response to current injection (PRE) and are potently inhibited by 100 nM tropisetron. After washout of tropisetron (WASH), the neurons recover. [Figure 9A] Figure 9 shows the activity of agonists on chimeric LGICs with the L131G mutation. A) Graph (logarithmic scale) of EC50 for L131 LBD mutants against known agonists normalized to unmodified α7-GlyR chimeric channels. [Figure 9B] Figure 9 shows the activity of agonists on chimeric LGICs with the L131G mutation. B) Graph of the EC50 of ACh and tropisetron for channels with mutations in the α7L131G-GlyR chimeric LGIC. [Figure 9C] Figure 9 shows the activity of agonists on a chimeric LGIC with the L131G mutation. C) Graph showing that the mutation resulting in a channel with high potency for varenicline and low potency for the endogenous ligand, acetylcholine (ACh), is optimal (gray shading). Unmod: unmodified α7-GlyR chimeric LGIC. [Figure 9D] Figure 9 shows the activity of agonists against chimeric LGICs with the L131G mutation. D) Graph showing activation of channels with the α7 L131G-GlyR mutation by Ach and varenicline after brief channel antagonism with picrotoxin (PTX). The solid line represents the period of molecule administration. [Figure 9E] Figure 9 shows the activity of agonists against a chimeric LGIC containing the L131G mutation. E) Action potentials of cortical neurons from mouse brains transduced with α7L131G, Q139L, Y217F-GlyR chimeric LGIC. The neurons fire in response to current injection (PRE) and are potently inhibited by 10 nM varenicline, even at over six-fold higher injected currents. After washout of varenicline (WASH), neuronal firing recovers. [Figure 10] Figure 10 includes graphs demonstrating that varenicline can activate chimeric receptors to induce behavioral effects in mice. In this case, expression of α7L131G, Q139L, Y217F-GlyR in unilateral VGAT-expressing neurons in the substantia nigra pars reticulata, followed by varenicline administration, induced contralateral rotation, consistent with varenicline-induced silencing of α7L131G, Q139L, Y217F-GlyR-expressing neurons. [Figure 11A] Figure 11 shows the chemical structures of exemplary LGIC agonists. A) Chemical structure of an LGIC agonist with a substitution pattern that is most compatible with the enhanced potency of α7Q79G, Y115F, G175K-GlyR. [Figure 11B] Figure 11 shows the chemical structures of exemplary LGIC agonists. B) Chemical structures of LGIC agonists with substitution patterns most compatible with the enhanced potency of α7L131G, Q139L, Y217F-GlyR, α7L131G, Q139L, Y217F-5HT3, or α7L131G, Q139L, Y217F-5HT3 HC. [Figure 11C]Figure 11 shows the chemical structures of exemplary LGIC agonists. C) Chemical structures of LGIC agonists with substitution patterns most compatible with the enhanced potency of α7L131G, Q139L, Y217F-GlyR or α7L131G, Q139L, Y217F-5HT3 HC. [Figure 12] Figure 12 shows chemogenetic perturbation of cortical neuron activity. A-B) Varenicline potently inhibited action potential firing in PSAM4-GlyR-expressing neurons by decreasing input resistance (A) and increasing rheobase (B). C) Cortical layer 2 / 3 neuron membrane properties were similar in PSAM4-GlyR-expressing neurons and mixed, untransfected control neurons. D-E) Varenicline depolarized (D) and induced firing (E) in PSAM4-5HT3 HC-expressing neurons. F) Cortical layer 2 / 3 neuron membrane properties were similar in PSAM4-5HT3 HC-expressing neurons and mixed, untransfected control neurons. Data are means ± SEM. Mann-Whitney U test, n / s P > 0.05, *** P < 0.001. [Figure 13A] Figure 13 shows silencing of PSAM4-GlyR neurons in mice. A) PSAM4-GlyR-IRES-EGFP was unilaterally targeted to the SNr. Inset: Schematic of unilateral SNr transmission resulting from silencing of the opposite rotation. Asterisk: nonspecific immunofluorescence. [Figure 13B] Figure 13 shows silencing of PSAM4-GlyR neurons in mice. B) Low doses of intraperitoneal varenicline induce contraversive rotations in mice expressing PSAM4-GlyR but not in sham-operated mice or mice expressing EGFP alone. [Figure 13C] Figure 13 shows silencing of PSAM4-GlyR neurons in mice. C) Two doses of varenicline 5 h apart gave similar rates of total turnover, indicating a lack of tachyphylaxis of the chemogenetic response. [Figure 13D]Figure 13 shows silencing of PSAM4-GlyR neurons in mice. D) Duration of chemogenetic silencing monitored by the time course of rotational response normalized to maximum rotation for each mouse. Pink (thin) arrow: amphetamine injection; cyan (thick) arrow: varenicline injection. Mann-Whitney U test, nsP>0.05, **P<0.01. [Figure 14-1] Figure 14 shows ultrapotent chemogenetic agonists (uPSEM). A) Comparison of uPSEM agonist EC50 at PSAM4 channels and endogenous varenicline targets with IC50 at α4β2 nAChRs at 1 μM ACh. LED: lowest effective dose in mice in SNr rotation assay. Unit: nM; brackets: SEM. Selectivity for PSAM4-GlyR in bold. [Figure 14-2] Figure 14 shows ultrapotent chemogenetic agonists (uPSEMs). B, C) Dose-response curves for PSAM4-GlyR, α4β2 nAChR, and 5HT3-R. uPSEM792 (B) is a 10% partial agonist of α4β2 nAChR, and uPSEM817 (C) inhibits α4β2 nAChR. [Figure 14-3] Figure 14 shows ultrapotent chemogenetic agonists (uPSEM). D) Current responses to uPSEM (2 μM) and Ach (10 μM) in HEK cells expressing α7 nAChRs. uPSEM does not activate α7 nAChRs, whereas Ach does. (E) Response amplitude normalized to ACh. [Figure 14-4] Figure 14 shows ultrapotent chemogenetic agonists (uPSEMs). F-J) uPSEM792, uPSEM793, uPSEM815, and uPSEM817 potently inhibit firing of PSAM4-GlyR-expressing cortical neurons by reducing the current required to fire an action potential (rheobase) (G-J). [Figure 14-5] Figure 14 shows ultrapotent chemogenetic agonists (uPSEMs). F-J) uPSEM792, uPSEM793, uPSEM815, and uPSEM817 potently inhibit firing of PSAM4-GlyR-expressing cortical neurons by reducing the current required to fire an action potential (rheobase) (G-J). [Figure 15A] Figure 15 shows the in vivo uPSEM dose response in mice unilaterally expressing PSAM4-GlyR in the SNr. Behavioral responses and time courses of uPSEM792 (A, B), uPSEM793 (C, D), uPSEM815 (E, F), and uPSEM817 (G, H). Time courses of rotational responses normalized to maximum rotation for each mouse. Pink (thin) arrows indicate amphetamine injection; blue (thick) arrows indicate uPSEM injection. [Figure 15B] See legend to Figure 15A. [Figure 15C] See legend to Figure 15A. [Figure 15D] See legend to Figure 15A. [Figure 15E] See legend to Figure 15A. [Figure 15F] See legend to Figure 15A. [Figure 15G] See legend to Figure 15A. [Figure 15H] See legend to Figure 15A. [Figure 16] Figure 16 shows an exemplary amino acid sequence of PSAM4-GlyR-KirM3M4 (SEQ ID NO: 13). PSAM4 mutations are highlighted. The sequence of the Kir2.1 (KCNJ2) endoplasmic reticulum (ER) transport sequence is underlined and shown in blue. [Figure 17] 17 shows an exemplary amino acid sequence of PSAM4-GlyR-B4sig (SEQ ID NO: 14). PSAM4 mutations are highlighted. The sequence of the β4 nAChR subunit (CHRNB4) signal sequence is underlined and shown in blue. [Figure 18]Figure 18 shows an exemplary amino acid sequence of PSAM4-GlyR-Kv2M3M4-soma (SEQ ID NO: 15). The PSAM4 mutation is highlighted. The sequence of the somatic targeting sequence of Kv2.1 (KCNB1) is underlined and shown in blue. [Figure 19A] Figure 19 shows an exemplary construct containing a nucleic acid sequence encoding a modified LGIC subunit. (A) Plasmid map of the AAV-Syn::PSAM4-GlyR-IRES-EGFP-WPRE construct containing a nucleic acid sequence encoding a modified LGIC subunit (SEQ ID NO: 33). [Figure 19B-1] Figure 19 shows an exemplary construct containing a nucleic acid sequence encoding a modified LGIC subunit. (B) Nucleic acid sequence of the AAV-Syn::PSAM4-GlyR-IRES-EGFP-WPRE construct containing a nucleic acid sequence encoding a modified LGIC subunit. [Figure 19B-2] This is a continuation of Figure 19B-1. [Figure 19B-3] This is a continuation of Figure 19B-2. [Figure 20A] Figure 20 shows an exemplary construct containing a nucleic acid sequence encoding a modified LGIC subunit. (A) Plasmid map of the AAV-CamkII::PSAM4-GlyR-IRES-EGFP-WPRE construct containing a nucleic acid sequence encoding a modified LGIC subunit (SEQ ID NO: 34). [Figure 20B-1] Figure 20 shows an exemplary construct comprising a nucleic acid sequence encoding a modified LGIC subunit. (B) Nucleic acid sequence of the AAV-CamkII::PSAM4-GlyR-IRES-EGFP-WPRE construct comprising a nucleic acid sequence encoding a modified LGIC subunit. [Figure 20B-2] This is a continuation of Figure 20B-1. [Figure 20B-3] This is a continuation of Figure 20B-2. DETAILED DESCRIPTION OF THE INVENTION

[0034] Detailed Description The present specification provides modified LGICs and methods for using the same. For example, the present specification provides modified LGICs comprising at least one modified LGIC subunit having an LBD and an IPD and at least one modified amino acid (e.g., an amino acid substitution). In certain cases, the modified LGIC may be a chimeric LGIC. For example, a chimeric LGIC may comprise an LBD from a first LGIC and an IPD from a second LGIC. In certain cases, the modified amino acid may confer pharmacological selectivity to the modified LGIC. For example, the modified amino acid may confer selective binding of an exogenous LGIC ligand to the modified LGIC. For example, the modified amino acid may confer reduced (e.g., minimized or eliminated) binding of unmodified LGIC subunits (e.g., LGIC subunits lacking the modification and / or endogenous LGIC subunits) to the modified LGIC. For example, the modified amino acid may confer reduced (e.g., minimized or eliminated) binding of an endogenous LGIC ligand to the modified LGIC.

[0035] The modified LGICs provided herein can be used, for example, in methods for treating channelopathies (e.g., nerve channelopathies or muscle channelopathies). For example, modified LGICs and exogenous LGIC ligands capable of binding to and activating the modified LGICs can be used to treat mammals with channelopathies. In certain cases, modified LGICs and exogenous LGIC ligands can be used to regulate (e.g., activate or inhibit) ion transport across the membrane of mammalian cells. In certain cases, modified LGICs and exogenous LGIC ligands can be used to regulate (e.g., increase or decrease) the excitability of cells in mammals.

[0036] Qualified LGIC As used herein, a "modified" LGIC is an LGIC containing at least one LGIC subunit. A modified LGIC can also refer to a pharmacologically selective actuator module (PSAM). A modified LGIC subunit can contain at least one modified amino acid (e.g., amino acid substitution) in the LBD and / or at least one modified amino acid (e.g., amino acid substitution) in the IPD. The modified LGIC subunits described herein can be modifications of LGICs from any appropriate species (e.g., human, rat, mouse, dog, cat, horse, cow, goat, pig, or monkey). In certain cases, a modified LGIC can include at least one chimeric LGIC subunit having a non-naturally occurring combination of an LBD from a first LGIC and an IPD from a second LGIC.

[0037] A modified LGIC (e.g., an LGIC comprising one or more modified LGIC subunits) can be a homomer (e.g., one having any number of the same modified LGIC subunits) or a heteromer (e.g., one having at least one modified LGIC subunit and any number of different LGIC subunits). In certain cases, the modified LGIC described herein can be a homomeric modified LGIC. The modified LGIC described herein can include any suitable number of modified LGIC subunits. In certain cases, the modified LGIC can be a trimer, tetramer, pentamer, or hexamer. For example, the modified LGIC described herein can be a pentamer.

[0038] The modified LGIC subunits described herein can be any suitable modification of LGIC. LGICs can conduct anions, cations, or both through the cell membrane in response to ligand binding. For example, LGICs can conduct sodium (Na) through the cell membrane in response to ligand binding. + ), potassium (K + ), calcium (Ca 2+ ), and / or chloride (Cl -) ions. Examples of LGICs include Cys-loop receptors (e.g., AChRs, such as nAChRs (e.g., muscle-type nAChRs or neuronal-type nAChRs)), gamma-aminobutyric acid (GABA; e.g., GABA A and GABA A These include, but are not limited to, -ρ (also known as GABAc) receptors, GlyRs, GluCl receptors, and 5HT3 receptors), ionotropic glutamate receptors (iGluRs, e.g., AMPA receptors, kainate receptors, NMDA receptors, and delta receptors), ATP-gated channels (e.g., P2X), and phosphatidylinositol 4,5-bisphosphate (PIP2)-gated channels. When the modified LGIC described herein is a chimeric LGIC, the chimeric LGIC can include an LBD selected from any suitable LGIC and an IPD selected from any suitable LGIC. When the LGIC includes multiple different subunits (e.g., when a neuronal nAChR includes α4, β2, and α7 subunits), the LBD and / or IPD can be selected from any subunit. For example, the LBD from an nAChR can be an α7 LBD. A representative rat α7 nAChR amino acid sequence (including both the LBD and IPD) is as follows: TIFF2025131833000011.tif68158

[0039] In certain cases, the modified LGIC subunits described herein may comprise an LBD derived from the α7 nAChR. Examples of α7 nAChR LBDs include, but are not limited to, the human α7 nAChR LBD having the amino acid sequence set forth in SEQ ID NO:1, the human α7 nAChR LBD having the amino acid sequence set forth in SEQ ID NO:2, and the human α7 nAChR LBD having the amino acid sequence set forth in SEQ ID NO:11. In certain cases, the α7 nAChR LBD may be a homolog, ortholog, or paralog of the human α7 nAChR LBD set forth in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:11. In certain cases, the α7 nAChR LBD has at least 75% sequence identity (e.g., at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97%, or at least 99% sequence identity) to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:11. may have: TIFF2025131833000012.tif118158

[0040] In certain cases, the modified LGIC subunit described herein may comprise an IPD derived from the 5HT3 receptor. Examples of 5HT3 IPDs include, but are not limited to, mouse 5HT3 IPD having the amino acid sequence set forth in SEQ ID NO:3 and human 5HT3 IPD having the amino acid sequence set forth in SEQ ID NO:4. In certain cases, the 5HT3 IPD may be a homolog, ortholog, or paralog of the 5HT3 IPD set forth in SEQ ID NO:3 or SEQ ID NO:4. In certain cases, the 5HT3 IPD may have at least 75% sequence identity (e.g., at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97%, or at least 99% sequence identity) to SEQ ID NO:3 or SEQ ID NO:4. TIFF2025131833000013.tif76158

[0041] In certain cases, the modified LGIC subunit described herein may comprise an IPD from a GlyR. An example of a GlyR IPD includes, but is not limited to, a mouse GlyR IPD having the amino acid sequence set forth in SEQ ID NO:5. In certain cases, the GlyR IPD may be a homolog, ortholog, or paralog of the human GlyR IPD set forth in SEQ ID NO:5. In certain cases, the GlyR IPD may have at least 75% sequence identity (e.g., at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97%, or at least 99% sequence identity) to SEQ ID NO:5. TIFF2025131833000014.tif33158

[0042] In certain cases, the modified LGIC subunits described herein are capable of binding to IPDs derived from GABA receptors (e.g., GABA A -ρ, also known as GABAc). A Examples of -ρ IPDs include, but are not limited to, human GABA receptor agonists having the amino acid sequence set forth in SEQ ID NO:9. A -ρ IPD. In certain cases, GABA A -ρ IPD is a human GABA A -ρ may be a homolog, ortholog, or paralog of an IPD. In certain cases, GABA A The -ρ IPD may have at least 75% sequence identity (e.g., at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97%, or at least 99% sequence identity) to SEQ ID NO:9. TIFF2025131833000015.tif32158

[0043] To calculate the percentage of sequence identity, two sequences are aligned and the number of identical amino acid residues between the two sequences is determined. The number of identical matches is divided by the length of the aligned region (i.e., the number of aligned amino acid residues) and multiplied by 100 to obtain the percentage of sequence identity. It is understood that the length of the aligned region can be a portion of one or both sequences up to the full length of the shortest sequence. It is also understood that a single sequence can be aligned with two or more other sequences and therefore have different percentage sequence identity values ​​across each aligned region. Aligning two or more sequences to determine the percentage of sequence identity can be performed using the computer program ClustalW with default parameters, which calculates the best match between a query and one or more subject sequences and aligns them so that identity, similarity, and difference can be determined. See, for example, Chenna et al., 2003, Nucleic Acids Res., 31(13):3497-500.

[0044] When the modified LGIC subunit described herein is a chimeric LGIC subunit, the chimeric LGIC subunit may contain an LBD and an IPD from the same species, or an LBD and an IPD from different species. In certain cases, the chimeric LGIC subunit may contain an LBD from a human LGIC protein and an IPD from a human LGIC protein. For example, the chimeric LGIC subunit may contain a human α7 LBD and a human GlyR IPD. In certain cases, the chimeric LGIC subunit may contain an LBD from a human LGIC protein and an IPD from a mouse LGIC protein. For example, the chimeric LGIC subunit may contain a human α7 LBD and a mouse 5HT3 IPD.

[0045] When the modified LGIC subunits described herein are chimeric LGIC subunits, the chimeric LGIC subunits can include various fusion points connecting the LBD and IPD, such that the number of amino acids in the LBD can vary when the LBD is fused to different IPDs to form chimeric channel subunits. For example, the length of the α7 nAChR LBD used to form a chimeric LGIC subunit with a 5HTS IPD is different from the length of the α7 nAChR LBD used to form a chimeric LGIC subunit with a GlyR IPD (e.g., compare Figures 1A and 1C with Figure 1B).

[0046] The modified LGIC subunits described herein can include an LBD with at least one modified amino acid and / or an IPD with at least one modified amino acid. For example, the modified LGIC subunits described herein can include an α7 LBD with at least 75% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 and an amino acid substitution at amino acid residues 27, 41, 77, 79, 131, 139, 141, 175, 210, 216, 217, and / or 219. For example, the modified LGIC subunits described herein can include a GlyR IPD with at least 75% sequence identity to the sequence set forth in SEQ ID NO:5 and an amino acid substitution at amino acid residue 298 of an α7-GlyR chimeric receptor (e.g., SEQ ID NO:7). For example, the modified LGIC subunit described herein may comprise a GABAC IPD having at least 75% sequence identity to SEQ ID NO:9 and an amino acid substitution at amino acid residue 298 of an α7-GABAc chimeric receptor (e.g., SEQ ID NO:10). In certain cases, the modified LGIC subunit described herein may contain more than one amino acid modification (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more). The modifications may be amino acid substitutions. In certain cases, the modified amino acid may confer pharmacological selectivity to the modified LGIC. For example, the modified amino acid may confer selective binding of an exogenous LGIC ligand to the modified LGIC. For example, the modified amino acid may result in reduced (minimized or eliminated) binding of an unmodified LGIC subunit (an LGIC subunit lacking the modification and / or an endogenous LGIC subunit) to the modified LGIC. For example, the modified amino acid may confer reduced (minimized or eliminated) binding of an endogenous LGIC ligand to the modified LGIC.

[0047] In certain embodiments, the modified LGIC subunits described herein may contain at least one modified amino acid that confers selective binding to an exogenous LGIC ligand (e.g., enhanced binding or enhanced efficacy) to the modified LGIC. Binding to the exogenous LGIC ligand may be more selective than binding to the endogenous LGIC ligand. A modified LGIC subunit with selective binding to an exogenous LGIC ligand may comprise any suitable LBD (e.g., α7 LBD). In certain embodiments, the modified LGIC subunit may comprise the α7 LBD set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12, and the amino acid modification may be a substitution at residues 77, 79, 131, 139, 141, 175, and / or 216. In certain cases, the tryptophan at amino acid residue 77 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with a hydrophobic amino acid residue, such as phenylalanine (e.g., W77F), tyrosine (e.g., W77Y), or methionine (e.g., W77M). For example, a modified LGIC subunit described herein can include an α7 LBD set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12, and having a W77F substitution. In certain cases, the glutamine at amino acid residue 79 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with an amino acid residue such as alanine (e.g., Q79A), glycine (e.g., Q79G), or serine (e.g., Q79S). For example, a modified LGIC subunit described herein can include an α7 LBD with a Q79G substitution. In certain cases, the leucine at amino acid residue 131 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with an amino acid residue such as alanine (e.g., L131A), glycine (e.g., L131G), methionine (e.g., L131M), asparagine (e.g., L131N), glutamine (e.g., L131Q), valine (e.g., L131V), or phenylalanine (e.g., L131F).In certain cases, the glycine at amino acid residue 175 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with an amino acid residue such as lysine (e.g., G175K), alanine (e.g., G175A), phenylalanine (e.g., G175F), histidine (e.g., G175H), methionine (e.g., G175M), arginine (e.g., G175R), serine (e.g., G175S), or valine (e.g., G175V). In certain cases, the proline at amino acid residue 216 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with an amino acid residue such as isoleucine (e.g., P216I). Modified LGIC subunits having selective binding to exogenous LGIC ligands can be substituted with any suitable IPD (e.g., GlyR IPD or GABA). A In certain embodiments, the modified LGIC subunit may comprise a GlyR IPD set forth in SEQ ID NO:5, where the amino acid modification may be a substitution at amino acid residue 298 of an α7-GlyR chimeric receptor (e.g., SEQ ID NO:7). In certain cases, the alanine at amino acid residue 298 of SEQ ID NO:7 can be substituted with an amino acid residue such as glycine (e.g., A298G). In certain embodiments, the modified LGIC subunit may comprise a GABA IPD set forth in SEQ ID NO:9. A -ρIPD, the amino acid modification of which may be α7-GABA A The substitution may be at amino acid residue 298 of the -ρ chimeric receptor (e.g., SEQ ID NO: 10). In certain cases, the tryptophan at amino acid residue 298 of SEQ ID NO: 10 can be substituted with an amino acid residue such as alanine (e.g., W298A).

[0048] In certain cases, the modified LGIC subunits described herein can contain two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more) amino acid modifications. For example, the modified LGIC subunits described herein can have at least 75 percent sequence identity to SEQ ID NO:7 and can contain a Q79G substitution and an A298G substitution. Further examples of modifications that can confer selective binding of an exogenous LGIC ligand to the modified LGIC include modifications described elsewhere (see, e.g., U.S. Patent No. 8,435,762).

[0049] Modified LGIC subunits that selectively bind (e.g., have enhanced binding or increased potency) to an exogenous LGIC ligand over an endogenous (e.g., canonical) LGIC ligand can also be described as having enhanced potency of the exogenous ligand. In certain cases, modified LGIC subunits described herein that selectively bind to an exogenous LGIC ligand can have at least a 4-fold (e.g., at least a 5-fold, at least a 6-fold, at least a 7-fold, at least a 8-fold, at least a 9-fold, at least a 10-fold, at least a 11-fold, at least a 12-fold, at least a 13-fold, at least a 14-fold, at least a 15-fold, at least a 16-fold, at least a 17-fold, at least a 18-fold, at least a 19-fold, or at least a 20-fold) enhanced potency for the exogenous ligand. In certain cases, modified LGIC subunits described herein that selectively bind to exogenous LGIC ligands can have enhanced potency for the exogenous ligand by about 4-fold to about 200-fold (e.g., about 4-fold to about 200-fold, about 5-fold to about 180-fold, about 6-fold to about 175-fold, about 7-fold to about 150-fold, about 8-fold to about 125-fold, about 9-fold to about 100-fold, about 10-fold to about 90-fold, about 11-fold to about 75-fold, about 12-fold to about 65-fold, about 13-fold to about 50-fold, about 14-fold to about 40-fold, or about 15-fold to about 30-fold). For example, modified LGIC subunits described herein that selectively bind to exogenous LGIC ligands can have enhanced potency for the exogenous ligand by about 10-fold to about 100-fold. For example, modified LGIC subunits described herein that selectively bind to exogenous LGIC ligands can have about 10-fold to about 20-fold enhanced potency with respect to the exogenous ligand.

[0050] In certain embodiments, the modified LGIC subunits described herein may contain at least one modified amino acid that confers reduced (e.g., minimized or eliminated) binding to the modified LGIC with unmodified LGIC subunits. Binding to modified LGIC subunits with the same modification may be more selective than binding to unmodified LGIC subunits. The unmodified LGIC subunit may be an LGIC subunit lacking a modification that confers reduced binding to the modified LGIC with unmodified LGIC subunits, or the unmodified LGIC may be an endogenous LGIC subunit. The modification that confers reduced binding to the modified LGIC with unmodified LGIC subunits may be a charge-reversal modification. The modified LGIC subunit with reduced binding to unmodified LGIC subunits may comprise any suitable LBD (e.g., α7 LBD). In certain embodiments, the modified LGIC subunit may comprise the α7 LBD set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12, and the amino acid modification may be a substitution at amino acid residues 27 and / or 41. For example, the arginine at amino acid residue 27 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 may be substituted with aspartic acid (e.g., R27D). For example, the glutamic acid at amino acid residue 41 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with arginine (e.g., E41R). In certain cases, the modified LGIC subunits described herein can include an α7 LBD with an R27D substitution and E41R.

[0051] In certain embodiments, the modified LGIC subunits described herein may contain at least one modified amino acid that confers reduced (e.g., minimized or eliminated) binding of an endogenous LGIC ligand to the modified LGIC. The endogenous LGIC ligand may be ACh. The modified LGIC subunit with reduced binding of an endogenous LGIC ligand may comprise any appropriate IPD (e.g., GlyR LBD). For example, the modified LGIC subunit may comprise the α7 LBD set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12, and the amino acid modification may be a substitution at amino acid residues 115, 131, 139, 210, 217, and / or 219. In certain cases, the tyrosine at amino acid residue 115 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 may be substituted with phenylalanine (e.g., Y115F). In certain cases, the leucine at amino acid residue 131 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with an amino acid residue such as alanine (e.g., L131A), glycine (e.g., L131G), methionine (e.g., L131M), asparagine (e.g., L131N), glutamine (e.g., L131Q), valine (e.g., L131V), or phenylalanine (e.g., L131F). In certain cases, the glutamine at amino acid residue 139 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with glycine (e.g., Q139G) or leucine (e.g., Q139L). In certain cases, the tyrosine at amino acid residue 210 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with phenylalanine (e.g., Y210F). In certain cases, the tyrosine at amino acid residue 217 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with phenylalanine (e.g., Y217F). In certain cases, the aspartic acid at amino acid residue 219 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with alanine (e.g., D219A).

[0052] In certain embodiments, the modified LGIC subunits described herein can contain at least one modified amino acid that confers increased ion conductance to the modified LGIC. In certain cases, the modified LGIC subunits can contain the 5HT3 IPD set forth in SEQ ID NO:3, where the amino acid modification can be a substitution at amino acid residues 425, 429, and / or 433. The modified LGIC subunits described herein can contain the 5HT3 IPD with an R425Q substitution, an R429D substitution, and an R433A substitution. In certain cases, the modified LGIC subunits can contain the 5HT3 IPD set forth in SEQ ID NO:4, where the amino acid modification can be a substitution at amino acid residues 420, 424, and / or 428. The modified LGIC subunits described herein can contain the 5HT3 IPD with an R420Q substitution, an R424D substitution, and an R428A substitution.

[0053] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-5HT3 LGIC subunit (SEQ ID NO: 6) having a human α7 nAChR LBD (SEQ ID NO: 1) with Q79G and Y115F amino acid substitutions, and a mouse 5HT3 IPD (SEQ ID NO: 3).

[0054] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-5HT3 LGIC subunit (SEQ ID NO: 6) having a human α7 nAChR LBD (SEQ ID NO: 1) with a Q79G amino acid substitution and a Q139G amino acid substitution, and a mouse 5HT3 IPD (SEQ ID NO: 3).

[0055] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with a Q79G and a Y115F amino acid substitution, and a human GlyR IPD (SEQ ID NO: 5) with an A298G amino acid substitution.

[0056] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with a Q79G and a Q139G amino acid substitution, and a human GlyR IPD (SEQ ID NO: 5) with an A298G amino acid substitution.

[0057] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with an R27D, E41R, Q79G, and Y115F amino acid substitution, and a human GlyR IPD (SEQ ID NO: 5) with an A298G amino acid substitution.

[0058] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with a substitution at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N), and a human GlyR IPD (SEQ ID NO: 5).

[0059] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions at amino acid residues 131 (e.g., L131G, L131A, L131M, or L131N) and Y115 (e.g., Y115F), and a human GlyR IPD (SEQ ID NO: 5).

[0060] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions at amino acid residues 131 (e.g., L131G, L131A, L131M, or L131N) and 139 (e.g., Q139L), and a human GlyR IPD (SEQ ID NO: 5).

[0061] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions at amino acid residues 131 (e.g., L131G, L131A, L131M, or L131N) and 217 (e.g., Y217F), and a human GlyR IPD (SEQ ID NO: 5).

[0062] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions at amino acid residues 131 (e.g., L131G, L131A, L131M, or L131N), 139 (e.g., Q139L), and 217 (e.g., Y217F), and a human GlyR IPD (SEQ ID NO: 5).

[0063] In certain cases, the modified LGIC described herein may include at least one chimeric α7-5HT3 LGIC subunit having a human α7 nAChR LBD (SEQ ID NO: 2) with a substitution at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) and a human 5HT3 IPD (SEQ ID NO: 4).

[0064] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with a substitution at amino acid residue 175 (e.g., G175K) and a human GlyR IPD (SEQ ID NO: 5).

[0065] In certain cases, the modified LGIC described herein may include at least one chimeric α7-5HT3 LGIC subunit having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions at amino acid residues 131 (e.g., L131G, L131A, L131M, or L131N) and 139 (e.g., Q139L), and a human 5HT3 IPD (SEQ ID NO: 4) with R420Q, R424D, and R428A substitutions.

[0066] In certain cases, the modified LGIC described herein may include at least one chimeric α7-5HT3 LGIC subunit having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions at amino acid residues 131 (e.g., L131G, L131A, L131M, or L131N), 139 (e.g., Q139L), and 217 (e.g., Y217F), and a human 5HT3 IPD (SEQ ID NO: 4) with R420Q, R424D, and R428A substitutions.

[0067] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions at amino acid residues 175 (e.g., G175K) and 115 (e.g., Y115F), and a human GlyR IPD (SEQ ID NO: 5).

[0068] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions at amino acid residues 175 (e.g., G175K), 115 (e.g., Y115F), and 79 (e.g., Q79G), and a human GlyR IPD (SEQ ID NO: 5).

[0069] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions at amino acid residues 175 (e.g., G175K), 77 (e.g., W77F), and 79 (e.g., Q79G), and a human GlyR IPD (SEQ ID NO: 5).

[0070] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with a substitution at amino acid residue 216 (e.g., P216I), and a human GlyR IPD (SEQ ID NO: 5).

[0071] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions at amino acid residues 216 (e.g., P216I) and 79 (e.g., Q79G), and a human GlyR IPD (SEQ ID NO: 5).

[0072] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 10) having a human α7 nAChR LBD (SEQ ID NO: 2) with a substitution at amino acid residue 131 (e.g., L131A, L131G, L131M, L131N, L131Q, L131V, or L131F), and a human GABAc IPD (SEQ ID NO: 9).

[0073] In certain cases, the modified LGIC described herein may include one or more additional polypeptide sequences. The polypeptide sequence may be a trafficking sequence (e.g., a trafficking (export) sequence and / or a signal sequence). Examples of trafficking (export) sequences include, but are not limited to, an ER trafficking sequence (e.g., FCYENEV (SEQ ID NO: 16)). Examples of signal sequences include, but are not limited to, a CHRNB4 signal sequence (e.g., TIFF2025131833000016.tif4128. The polypeptide sequence can be a targeting sequence. Examples of targeting sequences include, but are not limited to, KCNB1 somatic targeting sequences (e.g., TIFF2025131833000017.tif12158 is included. One or more additional polypeptide sequences can be included in the modified LGIC at any suitable position. In certain cases, the additional polypeptide sequence can be a terminal (e.g., C-terminal or N-terminal) polypeptide sequence. In certain cases, the additional polypeptide sequence can be an insertion. In certain cases, the additional polypeptide sequence can be a substitution.

[0074] In certain cases, the modified LGIC described herein can include a transport sequence. For example, the modified LGIC can include an ER transport sequence (e.g., FCYENEV (SEQ ID NO: 16)). An exemplary modified LGIC including an α7 nAChR LBD with an L131G substitution, a Q139L substitution, and a Y217F substitution (e.g., each of which is relative to the residue numbers shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12), and a GlyR IPD with an ER transport sequence inserted between residues 142 and 143 (e.g., relative to the residue numbers shown in SEQ ID NO: 5) is shown in Figure 16.

[0075] In certain cases, the modified LGICs described herein can include a signal sequence. For example, the modified LGIC can include a CHRNB4 signal sequence (e.g., An exemplary modified LGIC including a CHRNB4 signal sequence substituting residues 1-22, an α7 nAChR LBD with an L131G substitution, a Q139L substitution, and a Y217F substitution (e.g., each of which is relative to the residue numbers shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12), and a GlyR IPD is shown in FIG.

[0076] In certain cases, the modified LGICs described herein may include a targeting sequence. For example, the modified LGIC may include a KCNB1 somatic targeting sequence (e.g., TIFF2025131833000019.tif11158. An exemplary modified LGIC comprising an α7 nAChR LBD with an L131G substitution, a Q139L substitution, and a Y217F substitution (e.g., each of which is relative to the residue numbers set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12), and a GlyR IPD with a KCNB1 somatic targeting sequence inserted between residues 142 and 143 (e.g., relative to the residue numbers set forth in SEQ ID NO:5) is shown in Figure 18. When the LBD and / or IPD are homologs, orthologs, or paralogs of the sequences described herein (e.g., SEQ ID NOs:1-5 and / or 9), it is understood that references to particular modified amino acid residues can be shifted to the corresponding amino acids in the homologs, orthologs, or paralogs. For example, residues 425, 429, and 433 of mouse 5HT3 IPD set forth in SEQ ID NO:3 correspond to residues 420, 424, and 428 in human 5HT3 IPD set forth in SEQ ID NO:4, and the R425Q, R429D, and R433A substitutions in mouse 5HT3 IPD correspond to the R420Q, R424D, and R428A substitutions in human 5HT3 IPD.

[0077] Any method can be used to obtain the modified LGIC subunits described herein. In certain cases, peptide synthesis methods can be used to produce the modified LGIC subunits described herein. Examples of peptide synthesis methods include, but are not limited to, solution-phase peptide synthesis and solid-phase peptide synthesis. In certain cases, protein biosynthesis methods can be used to produce the modified LGIC subunits described herein. Examples of protein biosynthesis methods include, but are not limited to, transcription and / or translation of nucleic acids encoding the phosphomimetic peptides provided herein. Similar modified LGIC subunits (e.g., modified subunits having essentially the same modifications and / or essentially the same amino acid sequence) will self-assemble through interactions between the LBDs to form the modified LGIC.

[0078] The present specification also provides nucleic acids encoding the modified LGIC subunits described herein, as well as constructs for expressing nucleic acids encoding the modified LGIC subunits described herein (e.g., synthetic constructs such as plasmids, non-viral vectors, viral vectors (e.g., adeno-associated virus, herpes simplex virus, or lentiviral vectors, etc.)).

[0079] The nucleic acid sequence encoding the modified LGIC subunit described herein can encode any of the LGICs described herein. In certain cases, the nucleic acid sequence provided herein can encode the LBD from any of the LGICs described herein. In certain cases, the nucleic acid sequence provided herein can encode the IPD from any of the LGICs described herein. When the nucleic acid sequence provided herein encodes a chimeric LGIC, the chimeric LGIC can include an LBD selected from any suitable LGIC and an IPD selected from any suitable LGIC.

[0080] In certain cases, the nucleic acid sequence can encode an LGIC described herein. For example, the nucleic acid sequence can encode a nAChR (e.g., an α7 nAChR). A representative nucleic acid sequence encoding the rat α7 nAChR amino acid sequence (including both the LBD and IPD) is as follows:

[0081] TIFF2025131833000020.tif183158

[0082] In certain cases, the nucleic acid sequence encoding the modified LGIC subunit described herein may encode the LBD from the α7 nAChR. Examples of nucleic acid sequences encoding the α7 nAChR LBD include, but are not limited to, the nucleic acid sequence set forth in SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22. In certain cases, the nucleic acid sequence encoding the α7 nAChR LBD may have at least 75% sequence identity (e.g., at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97%, or at least 99% sequence identity) to SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22.

[0083] TIFF2025131833000021.tif126158TIFF2025131833000022.tif154158

[0084] In certain cases, the nucleic acid sequence encoding the modified LGIC subunit described herein may encode an IPD from the 5HT3 receptor. Examples of nucleic acid sequences encoding 5HT3 IPD include, but are not limited to, the nucleic acid sequence set forth in SEQ ID NO:23 and SEQ ID NO:24. In certain cases, the nucleic acid sequence encoding 5HT3 IPD may have at least 75% sequence identity (e.g., at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97%, or at least 99% sequence identity) to SEQ ID NO:23 or SEQ ID NO:24.

[0085] TIFF2025131833000023.tif191158

[0086] In certain cases, the nucleic acid sequence encoding the modified LGIC subunit described herein may encode an IPD from a GlyR. Examples of nucleic acid sequences encoding a GlyR IPD include, but are not limited to, the nucleic acid sequence set forth in SEQ ID NO:5. In certain cases, the GlyR IPD may be a homolog, ortholog, or paralog of the human GlyR IPD set forth in SEQ ID NO:5. In certain cases, the GlyR IPD IPD may have at least 75% sequence identity (e.g., at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97%, or at least 99% sequence identity) to SEQ ID NO:25.

[0087] TIFF2025131833000024.tif75158

[0088] In certain cases, the nucleic acid sequences encoding the modified LGIC subunits described herein are capable of binding to GABA receptors (e.g., GABA C Also known as GABA A-ρ , ) can encode IPD from GABA A-ρExamples of nucleic acid sequences encoding IPD include, but are not limited to, the nucleic acid sequence set forth in SEQ ID NO: 26. In particular, GABA A-ρ The IPD may have at least 75% sequence identity to SEQ ID NO:26 (e.g., at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97% or at least 99% sequence identity).

[0089] TIFF2025131833000025.tif75158

[0090] In calculating the percentage of sequence identity (%), two sequences are aligned and the number of identical amino acid residues between the two sequences is determined. The number of identical matches is divided by the length of the aligned region (i.e., the number of aligned nucleic acid residues) and multiplied by 100 to arrive at the percentage of sequence identity (%) value. It should be understood that the length of the aligned region can be a portion of one or both sequences up to the full length of the shortest sequence. It should also be understood that a single sequence can be aligned with multiple other sequences, and therefore can have different percentage of sequence identity (%) values ​​across each aligned region. The alignment of two or more sequences to determine the percentage of sequence identity (%) can be performed using the computer program ClustalW with default parameters, which calculates the best match between a query and one or more target sequences and aligns them so that identity, similarity, and difference can be determined. For example, see Chenna et al., 2003, Nucleic Acids Res., 31(13):3497-500.

[0091] A nucleic acid sequence encoding a modified LGIC described herein can contain at least one modified nucleic acid, such that the nucleic acid sequence can encode an LBD having at least one modified amino acid and / or an IPD having at least one modified amino acid. In certain cases, a nucleic acid sequence encoding a modified LGIC described herein can contain more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more) modified nucleic acids. For example, a nucleic acid sequence can encode a modified LGIC subunit containing an α7 LBD having at least 75% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 and amino acid substitutions at amino acid residues 27, 41, 77, 79, 131, 139, 141, 175, 210, 216, 217, and / or 219. Examples of nucleic acid codons at codon numbers 27, 41, 77, 79, 131, 139, 141, 175, 210, 216, 217, and / or 219 that can result in amino acid substitutions at amino acid residues 27, 41, 77, 79, 131, 139, 141, 175, 210, 216, 217, and / or 219 can be as shown below.

[0092] Table 12. Codon usage resulting in LBD amino acid substitutions TIFF2025131833000026.tif80163

[0093] For example, a nucleic acid sequence can encode a GlyR IPD having at least 75% sequence identity to the sequence set forth in SEQ ID NO:5, and a modified LGIC subunit comprising an amino acid substitution of α7-GlyR IPD at amino acid residue 298. An example of a nucleic acid codon at codon number 298 that can result in an amino acid substitution at amino acid residue 298 can be as shown below:

[0094] Table 13. Codon usage resulting in GlyR IPD amino acid substitutions TIFF2025131833000027.tif11163*Numbering is relative to the residue numbers listed in SEQ ID NO:7.

[0095] For example, the nucleic acid sequence can encode a GABAC IPD having at least 75% sequence identity to SEQ ID NO:9 and a modified LGIC subunit containing an amino acid substitution at amino acid residue 298 of the α7 GABAC chimeric receptor. An example of a nucleic acid codon at codon number 298 that can result in an amino acid substitution at amino acid residue 298 can be as shown below:

[0096] Table 14. Codon usage resulting in GABAC IPD amino acid substitutions TIFF2025131833000028.tif11163*Numbering is relative to the residue numbers set forth in SEQ ID NO:10.

[0097] In certain cases, a nucleic acid sequence encoding a modified LGIC described herein may encode an α7-5HT3 chimeric receptor (e.g., comprising a human α7 nAChR LBD (SEQ ID NO: 1) and a mouse 5HT3 IPD (SEQ ID NO: 3) component) as set forth in SEQ ID NO: 6. Examples of nucleic acid sequences encoding α7-5HT3 chimeric receptors comprising a human α7 nAChR LBD and a mouse 5HT3 IPD include, but are not limited to, the nucleic acid sequence set forth in SEQ ID NO: 27.

[0098] TIFF2025131833000029.tif169158

[0099] In certain cases, a nucleic acid sequence encoding a modified LGIC described herein may encode an α7-GlyR chimeric receptor (e.g., comprising a human α7 nAChR LBD (SEQ ID NO:2) and a human GlyR IPD (SEQ ID NO:5)) as set forth in SEQ ID NO:7. An example of a nucleic acid sequence encoding an α7-GlyR chimeric receptor comprising a human α7 nAChR LBD and a human GlyR IPD includes, but is not limited to, the nucleic acid sequence set forth in SEQ ID NO:28.

[0100] TIFF2025131833000030.tif154158

[0101] In certain cases, a nucleic acid sequence encoding a modified LGIC described herein may encode an α7-GABAC chimeric receptor (e.g., comprising a human α7 nAChR LBD (SEQ ID NO:2) and a human GABAC IPD (SEQ ID NO:9)) as set forth in SEQ ID NO:10. An example of a nucleic acid sequence encoding a chimeric receptor comprising a human α7 nAChR LBD (SEQ ID NO:2) and a human GABAC IPD (SEQ ID NO:9), comprising a human α7 nAChR LBD and a human GABAC IPD, includes, but is not limited to, the nucleic acid sequence set forth in SEQ ID NO:29.

[0102] TIFF2025131833000031.tif154158

[0103] In certain cases, the nucleic acid sequence encoding the modified LGIC described herein may include a nucleic acid sequence encoding one or more additional polypeptide sequences (e.g., a trafficking sequence, e.g., a trafficking (export) sequence and / or a signal sequence, or a targeting sequence). Examples of nucleic acid sequences encoding trafficking (export) sequences include, but are not limited to, nucleic acid sequences encoding ER trafficking sequences (e.g., TIFF2025131833000032.tif4128. Examples of nucleic acid sequences encoding signal sequences include, but are not limited to, nucleic acid sequences encoding the CHRNB4 signal sequence (e.g., Examples of nucleic acid sequences encoding targeting sequences include, but are not limited to, nucleic acid sequences encoding KCNB1 somatic targeting sequences (e.g., Contains TIFF2025131833000034.tif26159.

[0104] In certain cases, nucleic acids encoding modified LGIC subunits described herein can be linked (e.g., operably linked) to one or more regulatory elements. For example, nucleic acids encoding modified LGIC subunits described herein can be operably linked to any suitable promoter. The promoter can be a native (i.e., minimal) promoter or a composite promoter. The promoter can be a ubiquitous (i.e., constitutive) promoter or a regulated promoter (e.g., inducible, tissue-specific, cell type-specific (e.g., neuron-specific, muscle-specific, glial-specific), and neural subtype-specific). Examples of promoters that can be used to drive expression of nucleic acids encoding modified LGIC subunits described herein include, but are not limited to, synapsin (SYN), CAMKII, CMV, CAG, enolase, TRPV1, POMC, NPY, AGRP, MCH, and orexin promoters. In certain cases, nucleic acids encoding modified LGIC subunits described herein can be operably linked to a neuron-specific promoter.

[0105] When a nucleic acid encoding a modified LGIC subunit described herein is present in a construct, the construct can be any suitable construct. The construct can be a nucleic acid (e.g., DNA, RNA, or a combination thereof) construct. Examples of constructs include, but are not limited to, a plasmid, a non-viral vector, or a viral vector (e.g., an adeno-associated virus vector, a herpes simplex virus vector, or a lentiviral vector). In certain cases, a construct containing a nucleic acid encoding a modified LGIC subunit described herein can express the modified LGIC subunit. In certain cases, the construct can contain an internal ribosome entry site (IRES, e.g., a bicistronic IRES). In certain cases, the construct can contain a nucleic acid sequence encoding a detectable marker (e.g., a fluorescent polypeptide, e.g., green fluorescent polypeptide (GFP; e.g., enhanced GFP (EGFP))). In certain cases, the construct can contain a nucleic acid sequence that provides a selectable marker (e.g., an antibiotic resistance marker, e.g., ampicillin resistance) to the construct. An exemplary plasmid map and nucleic acid sequence of a construct comprising a nucleic acid sequence encoding a modified LGIC subunit described herein is shown in Figure 19. Another exemplary plasmid map and nucleic acid sequence of a construct comprising a nucleic acid sequence encoding a modified LGIC subunit described herein is shown in Figure 20.

[0106] The present specification also provides cells (e.g., mammalian cells) having the modified LGIC described herein. Mammalian cells having the modified LGIC described herein can be obtained by any suitable method. In certain cases, a pre-assembled modified LGIC can be provided to the cell. In certain cases, a nucleic acid encoding a modified LGIC subunit described herein can be provided to the cell under conditions in which the modified LGIC subunit is translated and under conditions in which multiple (e.g., 3, 4, 5, 6, or more) modified LGIC subunits can be assembled into the modified LGIC described herein.

[0107] LGIC Ligand The present specification also provides LGIC ligands that can bind to and activate the modified LGICs described herein. LGIC ligands can also be referred to as pharmacologically selective effector modules (PSEMs). LGIC ligands that can bind to and activate the modified LGICs described herein can be exogenous or endogenous. LGIC ligands that can bind to and activate the modified LGICs described herein can be naturally occurring or synthetic. LGIC ligands that can bind to and activate the modified LGICs described herein can be canonical or non-canonical. LGIC ligands that can bind to and activate the modified LGICs described herein can be agonists or antagonists. In certain cases, the LGIC ligand is an exogenous LGIC agonist. Examples of LGIC ligands include, but are not limited to, ACh, nicotine, epivastatin, cytisine, RS56812, tropisetron, nortopisetron, PNU-282987, PHA-543613, Compound 0353, Compound 0354, Compound 0436, Compound 0676, Compound 702, Compound 723, Compound 725, granisetron, ivermectin, mequitazine, promazine, varenicline, Compound 765, Compound 770, 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-dioxide, Compound 773, and Compound 774 (see, e.g., Figures 3B, 5C, 11A, 11B, and 11C).

[0108] An LGIC ligand that can bind to and activate a modified LGIC described herein may have selective binding (e.g., enhanced binding or increased potency) for the modified LGIC described herein (e.g., compared to unmodified LGIC). In certain cases, an LGIC ligand that can bind to and activate a modified LGIC described herein does not bind to and activate an endogenous receptor (e.g., endogenous LGIC). An LGIC ligand that selectively binds to and activates a modified LGIC described herein over an unmodified LGIC ligand (e.g., a modified LGIC having at least one amino acid modification that confers pharmacological selectivity to the modified LGIC) may be described as having enhanced potency for the modified LGIC. In certain cases, a modified LGIC subunit described herein that selectively binds to an exogenous LGIC ligand may have an enhanced potency of at least 5-fold (e.g., at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at least 95-fold, at least 100-fold, at least 125-fold, at least 150-fold, at least 200-fold, at least 250-fold, or at least 300-fold) relative to the modified LGIC. For example, an LGIC ligand that selectively binds to and activates a modified LGIC may have enhanced potency relative to the modified LGIC by about 10 to about 300 times (e.g., about 10 to about 250 times, about 10 to about 200 times, about 10 to about 150 times, about 10 to about 100 times, about 25 to about 300 times, about 50 to about 300 times, about 100 to about 300 times, about 200 to about 300 times, about 25 to about 250 times, about 50 to about 200 times, or about 100 to about 150 times). In certain cases, an LGIC ligand that binds to and activates a modified LGIC described herein may have a ligand potency of less than 25 nM (e.g., less than 22 nM, less than 20 nM, less than 17 nM, less than 15 nM, less than 13 nM, less than 12 nM, less than 11 nM, less than 10 nM, less than 5 nM, less than 2 nM, or less than 1 nM).For example, an LGIC ligand that binds to and activates a modified LGIC described herein may have a ligand potency of less than 15 nM. In certain cases, an LGIC ligand may have an EC50 of less than 25 nM (e.g., less than 22 nM, less than 20 nM, less than 17 nM, less than 15 nM, less than 13 nM, less than 12 nM, less than 11 nM, or less than 10 nM) for a modified LGIC subunit described herein. For example, an LGIC ligand (e.g., tropisetron) may bind to a modified LGIC subunit (e.g., α7) described herein. Q79G -GlyR A298G For example, an LGIC ligand (e.g., nortropisetron) may have an EC50 of about 11 nM for a modified LGIC subunit (e.g., α7) described herein. Q79G、Y115F -GlyR A298G ) of about 13 nM. In certain cases, an LGIC ligand may have an EC50 of greater than 20 μM (e.g., greater than 22 μM, greater than 25 μM, greater than 35 μM, greater than 50 μM, greater than 65 μM, greater than 80 μM, or greater than 100 μM) for a modified LGIC subunit described herein. For example, an LGIC ligand (e.g., ACh) may have an EC50 of greater than 20 μM (e.g., greater than 22 μM, greater than 25 μM, greater than 35 μM, greater than 50 μM, greater than 65 μM, greater than 80 μM, or greater than 100 μM) for a modified LGIC subunit described herein (e.g., α7 Q79G、Y115F -GlyR A298G ) may have an EC50 of greater than 100 μM.

[0109] In certain embodiments, the LGIC ligand can be a synthetic ligand capable of binding to and activating the modified LGIC described herein, and can be quinuclidine, tropane, 9-azabicyclo[3.3.1]nonane, or 2-phenyl-7,8,9,10-tetrahydro-6H-6,10-methanoazepino[4,5-g]quinoxaline.

[0110] LGIC ligands capable of binding to and activating the modified LGICs described herein have Formula I: TIFF2025131833000035.tif32128 (wherein X1 and X2 can independently be CH, CH2, O, NH, or NMe; each n can independently be 0 or 1; Y can be O or S; A can be an aromatic substituent; and R can be H or pyridinylmethylene). Examples of aromatic substituents include, but are not limited to, 4-chloro-benzene, 1H-indole, 4-(trifluoromethyl)benzene, 4-chlorobenzene, 2,5-dimethoxybenzene, 4-chloroaniline, aniline, 5-(trifluoromethyl)pyridin-2-yl, 6-(trifluoromethyl)nicotine, and 4-chloro-benzene.

[0111] An LGIC ligand capable of binding to and activating the modified LGIC described herein can be quinuclidine. Quinuclidine has the formula II: TIFF2025131833000036.tif18128 (wherein X3 can be O, NH, or CH2; Y can be O or S; A can be an aromatic substituent; and R can be H or pyridinylmethylene). The aromatic substituent may have the structure: Examples of aromatic substituents include, but are not limited to, 1H-indole, 4-(trifluoromethyl)benzene, 4-chlorobenzene, 2,5-dimethoxybenzene, 4-(trifluoromethyl)benzene, 4-chloroaniline, aniline, 5-(trifluoromethyl)pyridin-2-yl, 6-(trifluoromethyl)nicotine, 3-chloro-4-fluorobenzene, 4-chloro-benzene, and 1H-indole. Examples of quinuclidines include, but are not limited to, compounds PNU-282987, PHA-543613, 0456, 0434, 0436, 0354, 0353, 0295, 0296, and 0676 (see, for example, Figure 5C, Table 3, and Table 6).

[0112] An LGIC ligand capable of binding to and activating a modified LGIC described herein can be a tropane. The tropane has formula III: TIFF2025131833000037.tif29128 (wherein X2 can be NH or NMe; X3 can be O, NH, or CH2; Y can be O or S; A can be an aromatic substituent) Examples of aromatic substituents include, but are not limited to, 1H-indole, 7-methoxy-1H-indole, 7-methyl-1H-indole, 5-chloro-1H-indole, and 1H-indazole. Examples of tropanes include, but are not limited to, tropisetron, pseudotropisetron, nortropisetron, compound 737, and compound 745 (see, e.g., Figure 5C, Table 3, and Table 6).

[0113] An LGIC ligand capable of binding to and activating the modified LGIC described herein can be 9-azabicyclo[3.3.1]nonane, which has formula IV: TIFF2025131833000038.tif27128 (wherein X1 can be CH, X2 can be NH or NMe, X3 can be O, NH, or CH; Y can be O or S; A can be an aromatic substituent) The aromatic substituent may have the structure: An example, but is not limited to, 4-chloro-benzene. Examples of 9-azabicyclo[3.3.1]nonanes include, but are not limited to, Compound 0536, Compound 0749, Compound 0751, Compound 0760, and Compound 0763 (see, for example, Figure 5C, Table 3, and Table 6).

[0114] In certain cases, the LGIC ligand can be 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, represented by formula V: The compound may have the structure shown in TIFF2025131833000039.tif14128, where R is H, an aromatic substituent, a methyl-, ethyl-, or other aliphatic group, an alkoxy-, ethoxy-, propoxy-, and isopropoxy-containing group, or an amino-containing group; and R is H, an aliphatic substituent (e.g., methyl), or an aromatic substituent (e.g., phenyl). Examples of R groups include, but are not limited to, phenyl, 2-toluyl, 3-pyridyl, 4-pyridyl, imidazole, pyrrole, pyrazole, triazole, isoxazol-3-amine, trifluoromethyl, methoxy, N,N-dimethylamino, and N,N-diethylamino. In certain cases, R and R can be connected to form a ring. Examples of 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepines include, but are not limited to, varenicline, Compound 0765, Compound 0770, Compound 0780, Compound 0782, Compound 0785, Compound 0788, Compound 0782, Compound 0789, Compound 0791, Compound 0793, Compound 0794, Compound 0795, Compound 0798, Compound 0799, Compound 0800, Compound 0801, Compound 0802, Compound 0803, Compound 0804, Compound 0805, Compound 0807, Compound 0808, Compound 0812, Compound 0813, Compound 815, Compound 816, and Compound 817 (see, e.g., Figure 11A, Figure 14A, Table 3, Table 9, Table 10, and Table 11).

[0115] In certain cases, the LGIC ligand is 2-(pyridin-3-yl)-1,5,6,7,8,9-hexahydro-5,9-methanoimidazo[4',5':4,5]benzo[1,2-d]azepine: TIFF2025131833000040.tif12128. Examples of 2-(pyridin-3-yl)-1,5,6,7,8,9-hexahydro-5,9-methanoimidazo[4',5':4,5]benzo[1,2-d]azepines include compound 0786 (see, e.g., Tables 10 and 11).

[0116] In certain cases, the LGIC ligand can be 7,8,9,10-tetrahydro-1H-6,10-methanoazepino[4,5-g]quinoxalin-2(6H)-one, represented by formula VI: TIFF2025131833000041.tif14128, where R1 is H or an aliphatic substituent (e.g., methyl); R2 is H, an aliphatic substituent (e.g., methyl), or an aromatic substituent; and R3 is O or S. Examples of 7,8,9,10-tetrahydro-1H-6,10-methanoazepino[4,5-g]quinoxalin-2(6H)-ones include, but are not limited to, compound 0783, compound 0784, compound 0790, or compound 0792 (see, e.g., Figure 11B, Table 10, and Table 11). For example, LGIC can be It can be 7,8,9,10-tetrahydro-1H-6,10-methanoazepino[4,5-g]quinoxalin-2(6H)-one, which has the structure TIFF2025131833000042.tif17128.

[0117] In certain cases, the LGIC ligand can be 1,4-diazabicyclo[3.2.2]nonane, represented by formula VII: TIFF2025131833000043.tif25128 (in the formula, R=H, F, NO2) Examples of 1,4-diazabicyclo[3.2.2]nonanes include, but are not limited to, 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-dioxide, compound 0773, and compound 0774 (see, e.g., Figure 11C, Table 6, and Table 9).

[0118] How to use Also provided herein are methods using the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein. The LGIC ligands capable of binding to and activating the modified LGICs can be used to activate the modified LGICs with temporal and / or spatial control based on delivery of the ligand.

[0119] In certain embodiments, the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein can be used to identify ligands that selectively bind to the modified LGICs described herein. For example, such screening methods can include providing one or more candidate ligands to the modified LGIC described herein and detecting binding between the candidate ligands and the modified LGIC.

[0120] Any suitable method can be used to detect binding between a candidate ligand and a modified LGIC, and any suitable method can be used to detect activity of the modified LGIC. For example, the ability of a ligand to bind to and activate a modified LGIC can be measured by assays including, but not limited to, membrane potential (MP) assays (e.g., fluorescent MP assays), radioactive binding assays, and / or voltage clamp measurements of peak and sustained currents.

[0121] In certain embodiments, the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein can be used to treat mammals with channelopathies (e.g., nerve or muscle channelopathies). For example, a mammal with a channelopathic disorder can be treated by administering a modified LGIC described herein, followed by an LGIC ligand capable of binding to and activating the modified LGIC. For example, a mammal with a channelopathic disorder can be treated by administering a modified LGIC described herein (e.g., comprising at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 6) having a human α7 nAChR LBD (SEQ ID NO: 2) with an R27D, E41R, Q79G, and Y115F amino acid substitution, and a human GlyR IPD (SEQ ID NO: 5) with an A298G amino acid substitution) followed by tropisetron. For example, a mammal having a channelopathy can be treated by administering a modified LGIC described herein that includes a modified human α7 nAChR LBD (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12) with an L131 amino acid substitution (e.g., L131G, L131A, L131M, or L131N) and, optionally, a Q79S amino acid substitution, a Q139L amino acid substitution, and / or a Y217F amino acid substitution, followed by administration of varenicline, tropisetron, and / or Compound 765.

[0122] Any type of mammal can be treated using the modified LGICs described herein and LGIC ligands that can bind to and activate the modified LGICs described herein.For example, humans and other primates, such as monkeys, can be treated using the modified LGICs described herein and LGIC ligands that can bind to and activate the modified LGICs described herein.In certain cases, dogs, cats, horses, cows, pigs, sheep, rabbits, mice, and rats can be treated using the modified LGICs described herein and LGIC ligands that can bind to and activate the modified LGICs described herein.

[0123] Any suitable method can be used to identify mammals having and / or at risk of developing a channelopathy, for example, genetic testing can be used to identify mammals having and / or at risk of developing a channelopathy.

[0124] Once a mammal has been identified as having a channelopathy and / or at risk of developing a channelopathy, the mammal may be administered or instructed to self-administer a modified LGIC described herein, and then administered or instructed to self-administer an LGIC ligand capable of binding to and activating the modified LGIC described herein. The modified LGIC described herein and the LGIC ligand capable of binding to and activating the modified LGIC described herein can be administered together or separately.

[0125] When the materials and methods described herein are used to treat mammals with channelopathies and / or mammals at risk of developing channelopathies, the channelopathies can be any channelopathies. As used herein, a channelopathies can be any disease or disorder that is caused by abnormal ion channel function and / or abnormal ligand function, or that can be alleviated by regulated ion channel function and / or altered cellular ion flux (e.g., calcium ion flux). A channelopathies can be congenital or acquired. Examples of channelopathies include, but are not limited to, Bartter syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT), congenital hyperinsulinemia, cystic fibrosis, Dravet syndrome, transient ataxia, erythromelalgia, generalized epilepsy (e.g., with febrile seizures), familial hemiplegic migraine, fibromyalgia, hyperkalemic periodic paralysis, hypokalemic periodic paralysis, Lambert-Eaton myasthenic syndrome, long QT syndrome (e.g., Romano-Ward syndrome), short QT syndrome, malignant hyperthermia, mucolipidosis type IV, myasthenia gravis, myotonia congenita, neuromyelitis optica, neuromyotonia, nonsyndromic hearing loss, congenital paramyotonia, retinitis pigmentosa, Timothy syndrome, tinnitus, seizures, trigeminal neuralgia, and multiple sclerosis. Alternatively, or in addition, the materials and methods described herein can be used for other applications, including, but not limited to, pain treatment, cancer cell treatment, appetite control, spasticity treatment, muscular dystonia treatment, tremor treatment, and movement disorder treatment.

[0126] In certain cases, the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein can be used to regulate cellular activity. The cellular activity regulated using the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein can be any cellular activity. Examples of cellular activity include, but are not limited to, active transport (e.g., ion transport), passive transport, excitation, inhibition, ion flux (e.g., calcium ion flux), and exocytosis. Cellular activity can be increased or decreased. For example, the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein can be used to regulate (e.g., increase) ion transport across a cell's membrane. For example, the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein can be used to regulate (e.g., increase) cellular excitability.

[0127] The modified LGICs described herein and LGIC ligands that can bind to and activate the modified LGICs described herein can be used to regulate the activity of any type of cell in a mammal. The cell can be a neuron, a glial cell, a muscle cell, an immune cell (e.g., neutrophils, eosinophils, basophils, lymphocytes, and monocytes), an endocrine cell, or a stem cell (e.g., an embryonic stem cell). In certain cases, the cell can be an excitable cell. The cell can be in vivo or ex vivo.

[0128] The modified LGIC described herein can be administered by any suitable method. The modified LGIC can be administered as a modified LGIC subunit or as a pre-assembled modified LGIC. The modified LGIC can be administered as a nucleic acid encoding the modified LGIC. The modified LGIC can be administered as a nucleic acid encoding the modified LGIC subunit described herein. For example, the nucleic acid can be delivered as a naked nucleic acid or using any suitable vector (e.g., a recombinant vector). The vector can be a DNA-based vector, an RNA-based vector, or a combination thereof. The vector can express the nucleic acid in dividing or non-dividing cells. Examples of recombinant vectors include, but are not limited to, plasmids, viral vectors (e.g., retroviral vectors, adenoviral vectors, adeno-associated viral vectors, and herpes simplex vectors), cosmids, and artificial chromosomes (e.g., yeast artificial chromosomes or bacterial artificial chromosomes). In certain cases, the nucleic acid encoding the modified LGIC subunit described herein can be expressed by an adeno-associated viral vector.

[0129] The modified LGIC described herein can be detected by any suitable method (e.g., to confirm its presence in cells). In certain cases, an agent that selectively binds the modified LGIC can be used to detect the modified LGIC. Examples of agents that can be used to bind to the modified LGIC described herein include, but are not limited to, antibodies, proteins (e.g., bungarotoxin), and small molecule ligands (e.g., PET ligands). The agent that selectively binds to the modified LGIC can include a detectable label (e.g., a fluorescent label, a radioactive label, a positron-emitting label, and an enzyme label). Methods for detecting LGIC expression in cells can include fluorescence imaging, autoradiography, functional MRI, PET, and SPECT.

[0130] The modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein can be administered to a mammal having a channelopathy and / or at risk of developing a channelopathy as a combination therapy with one or more additional agents / therapies used to treat the channelopathy. For example, a combination therapy used to treat a mammal having a channelopathy as described herein can include administering a modified LGIC described herein and an LGIC ligand capable of binding to and activating the modified LGIC described herein, and treating with acetazolamide, dichlorophenamide, mexilitine, glucose, calcium gluconate, L-DOPA, muscle stimulation, spinal cord stimulation, brain stimulation, and / or neurostimulation.

[0131] In embodiments in which the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein are used in combination with additional agents / therapies used to treat channelopathies, the one or more additional agents can be administered simultaneously or independently. For example, the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein can be administered first, followed by the one or more additional agents, or vice versa. In embodiments in which the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein are used in combination with one or more additional therapies used to treat channelopathies, the one or more additional therapies can be administered simultaneously or independently with the administration of the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein. For example, the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein can be administered before, during, or after the administration of the one or more additional therapies.

[0132] In certain cases, the modified LGICs described herein and / or LGIC ligands capable of binding to and activating the modified LGICs described herein can be formulated into pharmaceutically acceptable compositions for administration to mammals having or at risk of developing a channelopathy. For example, a therapeutically effective amount of the modified LGICs described herein (e.g., nucleic acids encoding the modified LGICs described herein) and / or LGIC ligands capable of binding to and activating the modified LGICs described herein can be formulated together with one or more pharmaceutically acceptable carriers (excipients) and / or diluents. Pharmaceutical compositions can be formulated for administration in solid or liquid forms, including, but not limited to, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules.

[0133] Pharmaceutically acceptable carriers, fillers, and vehicles that can be used in the pharmaceutical compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphate, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0134] Pharmaceutical compositions containing the modified LGICs described herein and / or LGIC ligands capable of binding and activating the modified LGICs described herein can be designed for oral, parenteral (including subcutaneous, intracranial, intraarterial, intramuscular, intravenous, intracoronary, intradermal, or topical) or inhalation administration. When administered orally, pharmaceutical compositions containing a therapeutically effective amount of the modified LGICs described herein (e.g., nucleic acids encoding the modified LGICs described herein) and / or LGIC ligands capable of binding and activating the modified LGICs described herein can be in the form of pills, tablets, or capsules. Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. Inhalation compositions can be delivered using, for example, an inhaler, nebulizer, and / or dry powder inhaler. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0135] A pharmaceutically acceptable composition comprising a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and / or an LGIC ligand capable of binding to and activating a modified LGIC described herein can be administered locally or systemically. In certain cases, a composition comprising a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and / or an LGIC ligand capable of binding to and activating a modified LGIC described herein can be administered systemically to a mammal (e.g., a human) by intravenous or oral administration, or by inhalation. In certain cases, a composition comprising a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and / or an LGIC ligand capable of binding to and activating a modified LGIC described herein can be administered locally to a target tissue of a mammal (e.g., a human) by transdermal, subcutaneous, intramuscular, intracranial, or open surgical administration (e.g., injection).

[0136] The effective amount may vary depending on the severity of the channelopathy, the route of administration, the age and general health of the subject, the use of excipients, the possibility of co-administration with other therapeutic treatments such as the use of other drugs, and the judgment of the treating physician.

[0137] The frequency of administration can be any frequency that ameliorates the symptoms of the channelopathy without causing significant toxicity to the mammal. For example, the frequency of administration can be from about once per week to about three times per day, from about twice per month to about six times per day, or from about twice per week to about once per day. The frequency of administration can remain constant or can vary over the course of treatment. A course of treatment with a composition comprising a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and / or an LGIC ligand capable of binding to and activating a modified LGIC described herein can include a rest period. For example, a therapeutically effective amount of a composition comprising a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and / or an LGIC ligand capable of binding to and activating a modified LGIC described herein can be administered daily for two weeks, followed by a two-week rest period, and such a regimen can be repeated multiple times. As with the effective amount, various factors can affect the actual administration frequency used for a particular application. For example, the effective amount, the duration of treatment, the use of multiple therapeutic agents, the route of administration, and the severity of the channelopathy may require an increase or decrease in the administration frequency.

[0138] The effective period for administering a therapeutically effective amount of a composition comprising a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and / or a LGIC ligand capable of binding to and activating a modified LGIC described herein is any period that ameliorates symptoms of a channelopathy without causing significant toxicity to a mammal. For example, the effective period can vary from several days to several weeks, several months, or even several years. In certain cases, the effective period for treating a channelopathy can range from about one month to about 10 years. Multiple factors influence the actual effective period used for a particular treatment. For example, the effective period can vary depending on the frequency of administration, the effective amount, the use of multiple therapeutic agents, the route of administration, and the severity of the channelopathy being treated.

[0139] In certain instances, the progress of treatment and symptoms of a mammal being treated for a channelopathy can be monitored. Any suitable method can be used to monitor symptoms of a channelopathy.

[0140] The present invention is further described in the following examples, which do not limit the scope of the invention as defined in the claims. [Example]

[0141] Example 1: Potency-enhancing ligand-binding domain mutations A panel of 41 α7-5HT3 chimeric channels with mutated LBDs was screened against a panel of 51 clinically used drugs with chemical similarity to nicotinic receptor agonists. The mutations are at the residues highlighted in Figure 1. The screen identified a Gln in the α7 nAChR LBD that enhances potency against the known nAChR agonist tropisetron. 79 We identified mutations in the α7 nAChR (Figure 2). These mutations (Q79A, Q79G, Q79S) reduce the size of the amino acid side chain. Several mutant ion channel-ligand combinations provided up to a 12-fold improvement in potency (Table 1, Figure 3). Standard α7 nAChR agonists, ACh, nicotine, epibatidine, and the smoking cessation drug varenicline were not significantly affected by the Q79A, Q79G, or Q79S mutations. However, a subset of α7 nAChR agonists showed enhanced potency with some mutations. Cytisine, RS56812, tropisetron, nortropisetron, and PNU-282987 all exhibited potency enhancement with the α7 nAChR. Q79G Furthermore, nortropisetron and PNU-282987 each demonstrated significantly improved efficacy against α7 Q79A -5HT3 and α7 Q79Sshowed significantly enhanced potency for α7 agonists. In general, agonists based on quinuclidine or tropane pharmacophores with linked aromatic structures that interact with the complementary binding surface of the ligand-binding domain showed improved potency with Gln79 substitution with smaller amino acid residues Ala, Gly, or Ser. For most agonists, the α7 Q79G -5HT3 was the most preferred mutant chimeric lion channel.

[0142] Table 1. Potency of nAChR agonists against chimeric cation channels mutated at Gln79 in HEK cells. Mean EC50, SEM (μM) in parentheses. TIFF2025131833000044.tif54157

[0143] These mutant LBDs were used to generate α7-GlyR chimeric channels with up to six-fold enhanced potency for most of these ligands (Figure 4A). Similar to the α7-5HT3 mutations, these mutations at Gln79 did not significantly affect the potency of ACh, nicotine, epibatidine, varenicline, or cytisine. However, tropisetron, nortropisetron, and RS56812 significantly increased the potency of α7-GlyR chimeric channels. Q79G Similar to the LBD mutations for α7-5HT3, nortropisetron showed significantly enhanced potency against α7-GlyR. Q79A -GlyR, and PNU-282987 has significantly enhanced potency against α7 Q79S For most agonists, the potency at α7-GlyR was significantly enhanced. Q79G -GlyR was the most preferred mutant chimeric lion channel.

[0144] Another correlation observed in the small molecule screen was that mutations at Trp77 W77F -5HT3 (EC50: 1.2 μM), α7 W77Y -5HT3 (EC50: 1.1 μM), and α7 W77FThe drug granisetron exerted agonist activity at the α7-5HT3 or α7-GlyR receptors (EC50: 0.66 μM). Granisetron is a 5HT3 receptor antagonist, but it does not activate α7-5HT3 or α7-GlyR.

[0145] These results indicate that mutation of Q79 (to A, G, or S) in the α7 nAChR LBD enhanced binding of known LGIC ligands to modified LGIC.

[0146] Example 2: Potency-enhancing ion pore domain mutations α7-GlyR channels carrying previously established IPD mutations in full-length glycine receptor channels (T258S and A288G, GlyR numbering; equivalent to T268S and A298G for α7-GlyR numbering) were tested for enhanced potency for the allosteric agonist ivermectin. T268S Channels with α7-GlyR were found to have a substantially ligand-free open probability, making them unsuitable for ligand-controlled manipulation of cells. α7-GlyR was effective in enhancing ivermectin efficacy at the full-length glycine receptor. A298G Mutations at α7-GlyR resulted in modest changes in the open probability in the absence of ligand. Therefore, this channel was tested for activity against a panel of known agonists. α7-GlyR activity was measured for the standard agonists ACh, nicotine, and epibatidine, as well as for varenicline and tropisetron. A298G Agonist potency was not significantly enhanced in α7 nAChR agonists. A subset of α7 nAChR agonists showed modest, up to 4-fold increases in potency: RS56812, cytisine, PNU-282987, and nortropisetron were significantly more potent. Thus, the effect of the IPD A298G mutation improved ligand potency, but, depending on the ligand structure, was not as effective as mutations in the LBD.

[0147] The Q79G mutation in the LBD and the A298G IPD mutation for the α7-GlyR were examined (Table 2). Q79G -GlyR A298G resulted in a synergistic enhancement of potency for α7 nAChR agonists, demonstrating up to an 18-fold potency enhancement for α7-GlyRs. The enhancement from this double mutant channel was greater than that from the individual mutations for the agonists RS56812, tropisetron, nortropisetron, and PNU-282987. Further highlighting the unexpected structural sensitivity of this mutation combination, multiple agonists, including ACh, nicotine, epibatidine, varenicline, and cytisine, interacted with both α7-GlyRs and α7-GlyRs. Q79G -GlyR A298G Thus, the combination of the LBD mutation Q79G with the IPD mutation A298G resulted in a synergistic effect that significantly increased the potency of some, but not all, nicotinic agonists by approximately 10-20 fold.

[0148] Table 2: Potency of nAChR agonists against mutant chimeric chloride channels. Mean EC50 and SEM (μM) for agonist activity in HEK cells expressing chimeric channels. TIFF2025131833000045.tif59168

[0149] These results indicate that mutation of Q79 (to A, G, or S) in the α7 nAChR LBD and / or mutation of A298 (to G) in the GlyR IPD further enhanced selective binding of known LGIC ligands to the modified LGIC.

[0150] Example 3: Molecules that exhibit enhanced potency α7 Q79G -GlyR A298GBased on the structure-activity relationships of known agonists that showed enhanced potency at α7, we tested various synthetic molecules consisting of either quinuclidine, tropane, or 9-azabicyclo[3.3.1]nonane pharmacophores bearing one or more aromatic side-chain substituents. Additionally, we also tested the known α7 nAChR agonist PHA-543613 (Walker et al. 2006, Wishka et al. 2006). Q79G -GlyR A298G These molecules generally exhibited 10- to 100-fold enhanced potency (Table 3), suggesting that for these pharmacophores, a specific range of structural features contributes to the α7 Q79G -GlyR A298G This demonstrates that the efficacy of the steroids is consistent with improved efficacy.

[0151] These results indicate that the modified LGIC can be activated by synthetic quinuclidine- and tropane-containing LGIC ligands.

[0152] Table 3: Potency of compounds against chimeric channels. Mean EC50 and SEM in parentheses for agonist activity in HEK cells expressing chimeric channels (μM). Partial refers to partial agonist activity. TIFF2025131833000046.tif230125

[0153] Example 4: Mutations that reduce acetylcholine responsiveness The α7 nAChR is relatively insensitive to ACh compared with other nAChR isoforms, and potency-enhancing mutations for tropane and quinuclidine ligands did not substantially alter the potency of acetylcholine at these channels. Therefore, the chimeric channels were further modified to reduce the acetylcholine responsiveness of these channels. Additional LBD mutations, Y115F and Q139G, significantly reduced acetylcholine responsiveness to >100 μM in certain cases, and the α7 Q79G、Y115F -5HT3, α7 Q79G、Q139G -5HT3, α7 Q79G、Q139G -GlyR A298, α7 Q79G、Y115F -GlyR A298G For example, α7 only modestly reduced the potency of certain agonists. Q79G、Y115F -GlyR A298G has an EC50 of 13 nM for nortropisetron and over 100 μM for ACh (Table 4).

[0154] Table 4: Potency of nAChR agonists against mutant chimeric chloride channels with reduced acetylcholine responsiveness. Mean EC50 and SEM (μM) for activity in HEK cells expressing the chimeric channels. TIFF2025131833000047.tif48161

[0155] These results indicate that the Y115F and / or Q139G mutations in the α7 nAChR LBD reduced binding of the endogenous LGIC ligand Ach to modified LGIC.

[0156] Example 5: Mutations that reduce association with endogenous receptor subunits The assembly of the α7 nAChR is based on the association of five homomeric subunits through interactions between the LBDs (Celie et al., 2004 Neuron 41: 907-14). To minimize unwanted association with endogenous α7 nAChR subunits and / or undesired association of chimeric channels, we identified potential intersubunit bridges by examining the crystal structure of an acetylcholine-binding protein (ABP) and identifying nearby intersubunit residues with opposite charges that also share homologous ionizable amino acids in the α7 nAChR receptor LBD. Charge-reversal mutations (switching the acidic member of a potential salt bridge to a basic residue and its basic partner to an acidic residue) were designed to disrupt intersubunit interactions with unmodified subunits but preserve interactions between subunits with charge-reversal mutations (Figure 6A). Chimeric LGIC subunits with charge-reversal mutations were able to selectively associate with each other without interacting with unmodified channels, e.g., endogenous α7 nAChR. The R27D, E41R double mutation in the α7 nAChR LBD resulted in functional channels (Fig. 6B). Co-expression of these charge-reversal channels with α7-5HT3 channels containing the unmodified sequence demonstrated that the charge-reversal subunit did not co-immunoprecipitate with the unmodified channel (Fig. 6C). R27D、E41R、Q79G、Y115F -GlyR A298G Combining potency-enhancing mutations and acetylcholine-blocking mutations to obtain α- and β-agonists revealed that some agonists retained high potency relative to their cognate agonists (Table 4, right column).

[0157] These results indicate that the R27D and E41R mutations in the α7 nAChR LBD reduced the association of modified LGIC subunits with other modified and / or endogenous LGIC subunits.

[0158] Example 6: LBD mutations that increase ligand potency Gly of the α7 nAChR LBD in the α7-GlyR chimeric channel 175 and Pro 216Mutations in Gly were tested. 175 Mutation of α7 to Lys G175K -GlyR) showed increased potency against ACh (5-fold) (Table 5). G175K It was also found that for the α7-GlyR, nicotine potency was enhanced 10-fold compared to the unmodified α7-GlyR chimeric channel (Table 5). 216 Mutation of α7 to Ile P216I -GlyR) did not substantially alter ACh potency (Table 5). However, α7 P216I The α7-GlyR showed more than four-fold increased nicotine potency compared to the unmodified α7-GlyR (Table 5). G175K -GlyR and α7 P216I These potency-enhancing mutations in the α7-GlyR also affected the potency of several other α7-GlyR agonists by up to 30-fold (Table 5). G175K For α-GlyRs, a greater than 10-fold potency enhancement was observed compared to α7-GlyRs for the clinically used drugs tropisetron, varenicline, cytisine, granisetron, and epibatidine. P216I For -GlyR, the potency enhancement was approximately 3-fold (Table 5).

[0159] Table 5. Enhancement of agonist potency by G175K and P216I mutations in a7GlyR chimeric channels. Unit: μM. Brackets: SEM. TIFF2025131833000048.tif232113nd=Not decided

[0160] For use in organisms that produce ACh, it is important to reduce the endogenous ACh potency in these channels composed of the α7 nAChR LBD. The mutation G175K could be further combined with other mutations that reduce sensitivity to ACh, such as Y115F and Y210F. Y115F、G175KFor α-GlyR, high potency for agonists based on the tropane or quinuclidine core structure was observed for tropisetron, granisetron, nortropisetron, PNU-282987, and PHA-543613, with significantly reduced potency for varenicline and cytisine (Table 5). G175K、Y210F For -GlyR, potency for most agonists was significantly reduced, but enhanced potency for granisetron was observed (Table 5).

[0161] To develop a channel with reduced ACh responsiveness but high potency to other agonists, we investigated the α7 G175K The -GlyR was combined with additional mutations that increased the potency of certain agonists. Combination with W77F decreased ACh potency and α7 W77F、G175K The α7-GlyR showed increased potency over the α7-GlyR for granisetron, nortopisetron, and tropisetron, but not for PNU282-987, varenicline, cytisine, or PHA-543613 (Table 5). Combining G175K with Q79G reduced ACh potency and increased α7 Q79G、G175K The α7-GlyR showed increased potency for nortropisetron, PHA-543613, and tropisetron (Table 5). However, this increased potency was not observed for other agonists, such as PNU282-987 or varenicline. G175K、Q139L -GlyR decreased ACh potency and increased potency for nortropisetron and tropisetron (Table 5).

[0162] By incorporating mutations at W77F, Q79G, L141F, Y115F, G175K, and Y210F in various combinations, further reductions in ACh potency were achieved while maintaining high potency for synthetic agonists, including those based on the tropane and quinuclidine core structures. Q79G、Y115F、G175K -GlyR reduced ACh responsiveness while maintaining a strong response to tropisetron (Table 5). These mutations also Y115F、G175KResponses to other tropane and quinuclidine core structures, particularly quinuclidine thioureas 702 and 703, and tropane esters 723, 725, 726, 736, 737, 738, and 745, were also enhanced relative to α7-GlyR and relative to α7-5HT3 (representative of endogenous α7 nAChR activity) (Table 6). Q79G、Y115F、G175K -GlyR also showed high sensitivity to ivermectin (Table 5). W77F、Q79G、G175K α7-GlyR reduced ACh responsiveness while maintaining high potency responses to tropisetron and nortropisetron (Table 5). W77F、Q79G、G175K α7-GlyR also showed enhanced potency for additional tropane-based core structures, such as compounds 723 and 725, and the clinically used drugs mequitazine and promazine (Table 6). W77F、G175K、Y210F α7-GlyR reduced ACh responsiveness but significantly improved efficacy to granisetron (Table 5). L141F、Y115F、G175K α7-GlyR reduced ACh responsiveness while conferring sensitivity to granisetron (Table 5). Q79G、Q139L、G175K -GlyR reduced ACh responsiveness but exhibited a potent response to nortropisetron (Table 5).

[0163] Table 6. Potency enhancement of tropane, quinuclidine agonist, 9-azabicyclo[3.3.1]nonane agonist, diazabicyclo[3.2.2]nonane agonist, and promazine by G175K and P216I α7GlyR chimeric channels. Indole and indazole aromatic (A) substituents attached at the 3-position. Units: μM. TIFF2025131833000049.tif235139TIFF2025131833000050.tif235148nd=Not determined;Bracket:SEM

[0164] α7 G175K -GlyR and α7 P216Iα7-GlyR was compatible with the non-associating mutations R27D, E41R, and GlyR IPD mutation A298G, along with mutations at Q79G, Y115F, and G175K, which further enhanced ligand potency for granisetron, epibatidine, varenicline, cytisine, PNU-282987, tropisetron, nortropisetron, and PHA-543613 (Table 7). R27D、E41R、Q79G、Y115F、G175K Combination with non-associating mutations to form 702, 723, 725, and 726 further improved potency for 702, 723, 725, and 726, with low ACh responsiveness (Table 6).

[0165] Table 7. G175K and A298G mutations in α7GlyR chimeric channels and α7GABAc (GABA A Agonist potency enhancement by W298A at the -ρ (also called ρ) channel. Units: μM. TIFF2025131833000051.tif79157nd=Not determined;Bracket:SEM

[0166] α7 Y115F Gly in the α7 nAChR LBD in the α7-GlyR chimeric channel 175 Further amino acid substitutions at α7 also enhanced agonist potency. Y115F The potency of tropisetron in the -GlyR chimeric channel was enhanced with additional mutations, including G175A (7.1-fold), G175F (2-fold), G175H (2.3-fold), G175K (5.6-fold), G175M (2.6-fold), G175R (5.8-fold), G175S (9.3-fold), and G175V (16.7-fold).

[0167] Table 8. Agonist potency enhancement by G175 mutation in α7GlyR Y115F chimeric channels. Unit: μM. TIFF2025131833000052.tif49148nd=Not determined;Bracket:SEM

[0168] Leu to smaller amino acids 131Mutations in α7 were found to reduce the potency of the canonical agonists Ach and nicotine, while significantly increasing the potency of varenicline, tropisetron, and several other agonists. L131A -GlyR and α7 L131G α7-GlyR reduced ACh responsiveness (6-fold) and enhanced potency to varenicline (8-fold and 17-fold, respectively) and tropisetron (2.5-fold and 3.6-fold, respectively) (Table 9). L131G -5HT3 HC reduced ACh responsiveness (5-fold) and enhanced efficacy to varenicline (16-fold) and tropisetron (2.3-fold) (Figure 9A and Table 9). L131G、Q139L -GlyR and α7 L131G、Y217F α7-GlyRs showed a similar potency enhancement for varenicline relative to α7-GlyRs (21-fold), but also decreased ACh sensitivity (−11-fold and −13-fold, respectively). Q79S、L131G α7-GlyR further improved efficacy over α7-GlyR for varenicline (89-fold) and tropisetron (15-fold). L131G、Q139L、Y217F α7-GlyR showed the greatest improvement in potency over α7-GlyR for varenicline (387-fold) and also showed a decrease in ACh potency (13-fold) (Figure 9B and Table 9). L131G、Q139L、Y217F The α7-GlyR also showed extremely high potency for Compound 770 (0.001 μM), Compound 773 (0.00034 μM), and Compound 774 (0.00013 μM) (FIG. 11). Q79S、L131G、Q139L α7-GlyR also improved potency over α7-GlyR for varenicline (31-fold) and tropisetron (3-fold), but reduced ACh potency (9-fold) (Figure 9B and Table 9). L131M -GlyR, α7 L131Q -GlyR, and α7 L131V -GlyR reduced ACh potency but enhanced efficacy against tropisetron, nortropisetron, PHA-543613, and granisetron (Table 9). L131F-GlyR was found to substantially reduce ACh potency but did not improve potency for other agonists (Table 8). L131G -GABAc substantially reduced ACh potency but did not improve potency for other agonists (Table 9). L131G、Q139L、Y217F α7-5HT3 HC (Table 9) improved the efficacy of varenicline 131-fold over α7-5HT3 (Table 1). L131G、Q139L、Y217F -5HT3 HC also showed high potency for Compound 770 (0.007 μM), Compound 773 (0.002 μM), and Compound 774 (0.004 μM) (Table 8).

[0169] Table 9: Agonist potency enhancement by chimeric channels with the L131 mutation. Unit: μM. TIFF2025131833000053.tif235122nd=Not determined;Bracket:SEM

[0170] Example 7: Chimeric LGICs in neurons α7 Q79G -GlyR A298G or the α7 Q79G、Y115F、G175K Mouse cortical neurons were transduced with AAV or DNA plasmids containing nucleic acids encoding the -GlyR chimeric LGIC. Low concentrations of tropisetron (30 nM or 100 nM) were administered to the mouse cortical neurons. Neuronal activity was suppressed by the application of low concentrations of the agonist (Figures 7 and 8C).

[0171] α7 L131G、Q139L、Y217F Mouse cortical neurons were transfected with a DNA plasmid containing a nucleic acid encoding a -GlyR chimeric LGIC. A low concentration of varenicline (10 nM) was administered to the mouse cortical neurons. Neuronal activity was suppressed by the application of low concentrations of the agonist (Figure 9C).

[0172] These results indicate that modulated LGIC activity can be controlled in neurons using low concentrations of the LGIC ligands, tropisetron and varenicline.

[0173] Example 8: Varenicline and Varenicline Derivatives and Chimeric LGICs in Therapy The smoking cessation drug varenicline is a potent partial a4b2 agonist. It is also a moderate α7 nAChR agonist and a 5HT3 agonist. It has excellent brain penetration.

[0174] Chimera Channel α7 L131G, Q139L, Y217F -GlyR and α7 L131G, Q139L, Y217F The α7-5HT3 HC has enhanced binding capacity for the ligand varenicline compared to the unmodified chimeric channels, α7-GlyR and α7-5HT3. However, varenicline activates endogenous ion channels, such as α7 nAChR, α4β2 nAChR, and serotonin receptor 3 (5HT3-R), and it would be desirable to obtain varenicline derivatives with high potency at engineered channels but reduced potency at these endogenous targets.

[0175] Using the crystal structure of varenicline bound to acetylcholine-binding protein (AChP), a molecular contact was found to exist at V106, which is homologous to L131 in the α7 nAChR sequence. Mutation to L131G improved varenicline potency by 20-fold and reduced Ach potency by 5-fold (Figure 9A).

[0176] Further mutations to L131G were performed to decrease ACh potency and to improve varenicline potency. While the changes in potency were mostly related to both molecules, a subset of mutations selectively enhanced varenicline potency by 360-fold and reduced ACh potency by 20-fold (Figures 9B and 9C). The response to 10 nM varenicline compared to 300 μM ACh can be seen on the right (Figure 9D). These chimeric channels exhibit a characteristic slow activation that acts as a low-pass filter for transient fluctuations in ACh. Note also that despite the high potency of the channel, it does not possess any ligand-independent activity, as demonstrated by the brief application of the channel antagonist picrotoxin prior to the addition of these ligands. Ligand-independent activity is registered as an outward current in this trace. When the chimeric channels were expressed in cortical neurons, 10 nM varenicline induced ultrapotent neuronal silencing (Figure 9E).

[0177] In vivo activity was also extremely potent in unilateral substantia nigra (SNr) silencing experiments. Contralateral rotation of the silenced SNr was observed at doses above 0.1 milligrams / kg (mpk). A 10-fold improvement in varenicline's efficacy over anti-nicotine activity was observed (Figure 10).

[0178] Varenicline derivatives were designed to reduce or eliminate endogenous varenicline activity. The potency of varenicline and each varenicline derivative was determined for the various chimeric channels (Table 10).

[0179] Table 10: Potency (EC50, μM) of varenicline and varenicline derivatives at chimeric channels and comparison with agonist potency at 5HT3-R and α4β2 nAChR. TIFF2025131833000054.tif109163TIFF2025131833000055.tif225164nd=undetermined;brackets:SEM

[0180] Table 10 shows the α7 L131G、Q139L、Y217F -GlyR or α7 L131G、Q139L、Y217F Specific chemical structures of LGIC agonists with substitution patterns compatible with high potency for −5HT3 HC are shown.

[0181] All molecules in Table 10 show reduced sensitivity for α7-GlyR and α7-5HT3 (which serve as a proxy for α7 nAChR potency). Compounds 780, 783, 789, 790, 791, 792, 793, 795, 798, 802, 803, 804, 805, 807, 808, 812, and 813 show reduced sensitivity for α7 nAChR. L131G、Q139L、Y217F -GlyR or α7 L131G、Q139L、Y217F Compound 780, Compound 783, Compound 791, Compound 792, Compound 793, Compound 798, Compound 802, Compound 803, Compound 807, or Compound 808 exhibits a potency of less than 30 nM for either α7 L131G、Q139L、Y217F -GlyR or α7 L131G、Q139L、Y217F Compounds 792, 795, 802, and 808 exhibit potencies of less than 10 nM for either α7-5HT3 HC. L131G、Q139L、Y217F -GlyR or α7 L131G、Q139L、Y217F -5HT3 HC and potency of greater than 1 μM for 5HT3-R and greater than 10 μM for α4β2 nAChR.

[0182] We assessed chemogenetic perturbations of cortical neuron activity. Reducing input resistance and increasing rheobase stimulated PSAMs. 4 Varenicline strongly inhibited action potential firing in neurons expressing -GlyR. The membrane properties of cortical layer 2 / 3 neurons were similar to those of PSAM. 4 The effects of varenicline on PSAM-GlyR-expressing neurons and on the mixed untransfected control neurons were similar. 4 -Depolarizes and induces firing in neurons expressing 5HT3 HC. Cortical layer 2 / 3 neuron membrane properties are related to PSAM. 4-5HT3 HC-expressing neurons and mixed untransfected control neurons (see, e.g., Figure 12).

[0183] PSAM 4 Silencing of PSAM-GlyR neurons was assessed in mice. 4 -GlyR-IRES-EGFP targeted unilaterally to the SNr. Low-dose intraperitoneal varenicline inhibited PSAM. 4 Varenicline induces contraversive rotations in mice expressing -GlyR, but not in sham-operated mice or mice expressing EGFP alone. Two doses of varenicline, 5 hours apart, showed similar rates of total rotation, indicating a lack of tachyphylaxis in the chemogenetic response. Duration of chemogenetic silencing monitored by the time course of rotational response normalized to maximum rotation for each mouse. See, for example, Figure 13.

[0184] Ultrapotent chemogenetic agonists were evaluated. uPSEM agonist EC50 was calculated using the PSAM 4 Channel and endogenous varenicline targets, as well as 1 μM ACh and α4β2 nAChRs were compared at IC50. 792 α4β2 nAChR and uPSEM 817 It was a 10% partial agonist of α4β2 nAChR and inhibited α4β2 nAChR. 792 , uPSEM 793 , uPSEM 815 , and uPSEM 817 reduces the current required to fire an action potential (rheobase) 4 -GlyR-expressing cortical neurons. See, e.g., Figure 14.

[0185] PSAM in SNr 4 The in vivo uPSEM dose response was evaluated in mice unilaterally expressing -GlyR. See, e.g., Figure 15.

[0186] Varenicline derivatives were designed to reduce or eliminate endogenous varenicline activity. The potency of varenicline and each varenicline derivative was determined for the various chimeric channels (Table 11).

[0187] Table 11. EC50 of varenicline and derivatives at chimeric channels with comparison of agonist potency at 5HT3-R and α4β2 nAChR MP (μM). In vivo potency determined in SNr silencing turnover assay in mice. nd: not determined. nr: no response. Values ​​are mean±SEM. TIFF2025131833000056.tif45161

[0188] Ethoxy and propoxy compounds (uPSEM) 815 and 817 are PSAMs 4 -GlyR. 817 The agonist has excellent selectivity and PSAM 4 uPSEM has 5,000- to 10,000-fold selectivity for α-GlyR over α7-GlyR, α7-5HT3, and 5HT3-R. 815 and uPSEM 817 showed no obvious α4β2 nAChR agonism up to 30 μM, and uPSEM 815 is PSAM from 5HT3-R 4 -GlyR has a selectivity of over 2000-fold.

[0189] Example 9: Chimeric LGICs in therapy Chemical genetic tools offer an attractive strategy for combining drug therapy with gene therapy. This is because the use of exogenously delivered ion channels selectively coupled to drug administration allows the same ion channel and ligand to be used to modulate cellular function in a consistent manner across different cell types in a variety of indications. The identification of ion channels that are well tolerated and gated by clinically used drugs is particularly attractive for potentially extending chemical genetics to human therapeutic applications.

[0190] For the drug tropisetron, we found that it inhibits the α7 receptor with an EC50 of 11 nM, similar to the reported IC50 of 10 nM tropisetron for its therapeutic target, the 5HT3 receptor. Q79G -GlyR A298G (Combrink et al. 2009 Pharmacological reports: PR 61: 785-97).

[0191] Other embodiments While the present disclosure has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative, and not limiting, of the scope of the disclosure, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the claims.

[0192] Sequence information SEQUENCE LISTING <110> Howard Hughes Medical Institute <120> MODIFIED LIGAND-GATED ION CHANNELS AND METHODS OF USE <150> US 62 / 584,428 <151> 2017-11-10 <150> US 62 / 729,716 <151> 2018-09-11 <160> 34 <170> PatentIn version 3.5 <210> 1 <211> 224 <212> PRT <213> human <400> 1 Met Arg Cys Ser Pro Gly Gly Val Trp Leu Ala Leu Ala Ala Ser Leu 1 5 10 15 Leu His Val Ser Leu Gln Gly Glu Phe Gln Arg Lys Leu Tyr Lys Glu 20 25 30 Leu Val Lys Asn Tyr Asn Pro Leu Glu Arg Pro Val Ala Asn Asp Ser 35 40 45 Gln Pro Leu Thr Val Tyr Phe Ser Leu Ser Leu Leu Gln Ile Met Asp 50 55 60 Val Asp Glu Lys Asn Gln Val Leu Thr Thr Asn Ile Trp Leu Gln Met 65 70 75 80 Ser Trp Thr Asp His Tyr Leu Gln Trp Asn Val Ser Glu Tyr Pro Gly 85 90 95 Val Lys Thr Val Arg Phe Pro Asp Gly Gln Ile Trp Lys Pro Asp Ile 100 105 110 Leu Leu Tyr Asn Ser Ala Asp Glu Arg Phe Asp Ala Thr Phe His Thr 115 120 125 Asn Val Leu Val Asn Ser Ser Gly His Cys Gln Tyr Leu Pro Pro Gly 130 135 140 Ile Phe Lys Ser Ser Cys Tyr Ile Asp Val Arg Trp Phe Pro Phe Asp 145 150 155 160 Val Gln His Cys Lys Leu Lys Phe Gly Ser Trp Ser Tyr Gly Gly Trp 165 170 175 Ser Leu Asp Leu Gln Met Gln Glu Ala Asp Ile Ser Gly Tyr Ile Pro 180 185 190 Asn Gly Glu Trp Asp Leu Val Gly Ile Pro Gly Lys Arg Ser Glu Arg 195 200 205 Phe Tyr Glu Cys Cys Lys Glu Pro Tyr Pro Asp Val Thr Phe Thr Val 210 215 220 <210> 2 <211> 229 <212> PRT <213> human <400> 2 Met Arg Cys Ser Pro Gly Gly Val Trp Leu Ala Leu Ala Ala Ser Leu 1 5 10 15 Leu His Val Ser Leu Gln Gly Glu Phe Gln Arg Lys Leu Tyr Lys Glu 20 25 30 Leu Val Lys Asn Tyr Asn Pro Leu Glu Arg Pro Val Ala Asn Asp Ser 35 40 45 Gln Pro Leu Thr Val Tyr Phe Ser Leu Ser Leu Leu Gln Ile Met Asp 50 55 60 Val Asp Glu Lys Asn Gln Val Leu Thr Thr Asn Ile Trp Leu Gln Met 65 70 75 80 Ser Trp Thr Asp His Tyr Leu Gln Trp Asn Val Ser Glu Tyr Pro Gly 85 90 95 Val Lys Thr Val Arg Phe Pro Asp Gly Gln Ile Trp Lys Pro Asp Ile 100 105 110 Leu Leu Tyr Asn Ser Ala Asp Glu Arg Phe Asp Ala Thr Phe His Thr 115 120 125 Asn Val Leu Val Asn Ser Ser Gly His Cys Gln Tyr Leu Pro Pro Gly 130 135 140 Ile Phe Lys Ser Ser Cys Tyr Ile Asp Val Arg Trp Phe Pro Phe Asp 145 150 155 160 Val Gln His Cys Lys Leu Lys Phe Gly Ser Trp Ser Tyr Gly Gly Trp 165 170 175 Ser Leu Asp Leu Gln Met Gln Glu Ala Asp Ile Ser Gly Tyr Ile Pro 180 185 190 Asn Gly Glu Trp Asp Leu Val Gly Ile Pro Gly Lys Arg Ser Glu Arg 195 200 205 Phe Tyr Glu Cys Cys Lys Glu Pro Tyr Pro Asp Val Thr Phe Thr Val 210 215 220 Thr Met Arg Arg Arg 225 <210> 3 <211> 247 <212> PRT <213> mouse <400> 3 Ile Ile Arg Arg Arg Pro Leu Phe Tyr Ala Val Ser Leu Leu Leu Pro 1 5 10 15 Ser Ile Phe Leu Met Val Val Asp Ile Val Gly Phe Cys Leu Pro Pro 20 25 30 Asp Ser Gly Glu Arg Val Ser Phe Lys Ile Thr Leu Leu Leu Gly Tyr 35 40 45 Ser Val Phe Leu Ile Ile Val Ser Asp Thr Leu Pro Ala Thr Ile Gly 50 55 60 Thr Pro Leu Ile Gly Val Tyr Phe Val Val Cys Met Ala Leu Leu Val 65 70 75 80 Ile Ser Leu Ala Glu Thr Ile Phe Ile Val Arg Leu Val His Lys Gln 85 90 95 Asp Leu Gln Arg Pro Val Pro Asp Trp Leu Arg His Leu Val Leu Asp 100 105 110 Arg Ile Ala Trp Ile Leu Cys Leu Gly Glu Gln Pro Met Ala His Arg 115 120 125 Pro Pro Ala Thr Phe Gln Ala Asn Lys Thr Asp Asp Cys Ser Gly Ser 130 135 140 Asp Leu Leu Pro Ala Met Gly Asn His Cys Ser His Val Gly Gly Pro 145 150 155 160 Gln Asp Leu Glu Lys Thr Pro Arg Gly Arg Gly Ser Pro Leu Pro Pro 165 170 175 Pro Arg Glu Ala Ser Leu Ala Val Arg Gly Leu Leu Gln Glu Leu Ser 180 185 190 Ser Ile Arg His Phe Leu Glu Lys Arg Asp Glu Met Arg Glu Val Ala 195 200 205 Arg Asp Trp Leu Arg Val Gly Tyr Val Leu Asp Arg Leu Leu Phe Arg 210 215 220 Ile Tyr Leu Leu Ala Val Leu Ala Tyr Ser Ile Thr Leu Val Thr Leu 225 230 235 240 Trp Ser Ile Trp His Tyr Ser 245 <210> 4 <211> 242 <212> PRT <213> Humana <400> 4 Ile Ile Arg Arg Arg Pro Leu Phe Tyr Val Val Ser Leu Leu Leu Pro 1 5 10 15 Ser Ile Phe Leu Met Val Met Asp Ile Val Gly Phe Tyr Leu Pro Pro 20 25 30 Asn Ser Gly Glu Arg Val Ser Phe Lys Ile Thr Leu Leu Leu Gly Tyr 35 40 45 Ser Val Phe Leu Ile Ile Val Ser Asp Thr Leu Pro Ala Thr Ala Ile 50 55 60 Gly Thr Pro Leu Ile Gly Val Tyr Phe Val Val Cys Met Ala Leu Leu 65 70 75 80 Val Ile Ser Leu Ala Glu Thr Ile Phe Ile Val Arg Leu Val His Lys 85 90 95 Gln Asp Leu Gln Gln Pro Val Pro Ala Trp Leu Arg His Leu Val Leu 100 105 110 Glu Arg Ile Ala Trp Leu Leu Cys Leu Arg Glu Gln Ser Thr Ser Gln 115 120 125 Arg Pro Pro Ala Thr Ser Gln Ala Thr Lys Thr Asp Asp Cys Ser Ala 130 135 140 Met Gly Asn His Cys Ser His Met Gly Gly Pro Gln Asp Phe Glu Lys 145 150 155 160 Ser Pro Arg Asp Arg Cys Ser Pro Pro Pro Pro Pro Arg Glu Ala Ser 165 170 175 Leu Ala Val Cys Gly Leu Leu Gln Glu Leu Ser Ser Ile Arg Gln Phe 180 185 190 Leu Glu Lys Arg Asp Glu Ile Arg Glu Val Ala Arg Asp Trp Leu Arg 195 200 205 Val Gly Ser Val Leu Asp Lys Leu Leu Phe His Ile Tyr Leu Leu Ala 210 215 220 Val Leu Ala Tyr Ser Ile Thr Leu Val Met Leu Trp Ser Ile Trp Gln 225 230 235 240 Tyr Ala <210> 5 <211> 202 <212> PRT <213> Humana <400> 5 Met Gly Tyr Tyr Leu Ile Gln Met Tyr Ile Pro Ser Leu Leu Ile Val 1 5 10 15 Ile Leu Ser Trp Ile Ser Phe Trp Ile Asn Met Asp Ala Ala Pro Ala 20 25 30 Arg Val Gly Leu Gly Ile Thr Thr Val Leu Thr Met Thr Thr Gln Ser 35 40 45 Ser Gly Ser Arg Ala Ser Leu Pro Lys Val Ser Tyr Val Lys Ala Ile 50 55 60 Asp Ile Trp Met Ala Cys Leu Leu Phe Val Phe Ser Ala Leu Leu 65 70 75 80 Glu Tyr Ala Ala Val Asn Phe Val Ser Arg Gln His Lys Glu Leu Leu 85 90 95 Arg Phe Arg Arg Lys Arg Arg His His Lys Glu Asp Glu Ala Gly Glu 100 105 110 Gly Arg Phe Asn Phe Ser Ala Tyr Gly Met Gly Pro Ala Cys Leu Gln 115 120 125 Only Lys Asp Gly Served Val Lys Gly Only Asn Asn Served Asn Thr Thr 130 135 140 Asn Pro Pro Pro Ala Pro Ser Lys Ser Pro Glu Glu Met Arg Lys Leu 145 150 155 160 Phe and Gln Arg with Lys Lys and Asp Lys with Ser Arg and Gly Phe 165 170 175 Pro Met Ala Phe Leu Ile Phe Asn Met Phe Tyr Trp Ile Ile Tyr Lys 180 185 190 Ile Will Arg Arg Glu Asp Will His Asn Gln 195,200 <210> 6 <211> 471 <212> PRT <213> artificial <220> <223> alpha7-5HT3 chimeric receptor sequence <400> 6 Met Arg Cys Ser Pro Gly Gly Val Trp Leu Ala Leu Ala Ala Ser Leu 1 5 10 15 Leu His Val Ser Leu Gln Gly Glu Phe Gln Arg Lys Leu Tyr Lys Glu 20 25 30 Leu Val Lys Asn Tyr Asn Pro Leu Glu Arg Pro Val Ala Asn Asp Ser 35 40 45 Gln Pro Leu Thr Val Tyr Phe Ser Leu Ser Leu Leu Gln Ile Met Asp 50 55 60 Val Asp Glu Lys Asn Gln Val Leu Thr Thr Asn Ile Trp Leu Gln Met 65 70 75 80 Ser Trp Thr Asp His Tyr Leu Gln Trp Asn Val Ser Glu Tyr Pro Gly 85 90 95 Val Lys Thr Val Arg Phe Pro Asp Gly Gln Ile Trp Lys Pro Asp Ile 100 105 110 Leu Leu Tyr Asn Ser Ala Asp Glu Arg Phe Asp Ala Thr Phe His Thr 115 120 125 Asn Val Leu Val Asn Ser Ser Gly His Cys Gln Tyr Leu Pro Pro Gly 130 135 140 Ile Phe Lys Ser Ser Cys Tyr Ile Asp Val Arg Trp Phe Pro Phe Asp 145 150 155 160 Val Gln His Cys Lys Leu Lys Phe Gly Ser Trp Ser Tyr Gly Gly Trp 165 170 175 Ser Leu Asp Leu Gln Met Gln Glu Ala Asp Ile Ser Gly Tyr Ile Pro 180 185 190 Asn Gly Glu Trp Asp Leu Val Gly Ile Pro Gly Lys Arg Ser Glu Arg 195 200 205 Phe Tyr Glu Cys Cys Lys Glu Pro Tyr Pro Asp Val Thr Phe Thr Val 210 215 220 Ile Ile Arg Arg Arg Pro Leu Phe Tyr Ala Val Ser Leu Leu Leu Pro 225 230 235 240 Ser Ile Phe Leu Met Val Val Asp Ile Val Gly Phe Cys Leu Pro Pro 245 250 255 Asp Ser Gly Glu Arg Val Ser Phe Lys Ile Thr Leu Leu Leu Gly Tyr 260 265 270 Ser Val Phe Leu Ile Ile Val Ser Asp Thr Leu Pro Ala Thr Ile Gly 275 280 285 Thr Pro Leu Ile Gly Val Tyr Phe Val Val Cys Met Ala Leu Leu Val 290 295 300 Ile Ser Leu Ala Glu Thr Ile Phe Ile Val Arg Leu Val His Lys Gln 305 310 315 320 Asp Leu Gln Arg Pro Val Pro Asp Trp Leu Arg His Leu Val Leu Asp 325 330 335 Arg Ile Ala Trp Ile Leu Cys Leu Gly Glu Gln Pro Met Ala His Arg 340 345 350 Pro Pro Ala Thr Phe Gln Ala Asn Lys Thr Asp Asp Cys Ser Gly Ser 355 360 365 Asp Leu Leu Pro Ala Met Gly Asn His Cys Ser His Val Gly Gly Pro 370 375 380 Gln Asp Leu Glu Lys Thr Pro Arg Gly Arg Gly Ser Pro Leu Pro Pro 385 390 395 400 Pro Arg Glu Ala Ser Leu Ala Val Arg Gly Leu Leu Gln Glu Leu Ser 405 410 415 Ser Ile Arg His Phe Leu Glu Lys Arg Asp Glu Met Arg Glu Val Ala 420 425 430 Arg Asp Trp Leu Arg Val Gly Tyr Val Leu Asp Arg Leu Leu Phe Arg 435 440 445 Ile Tyr Leu Leu Ala Val Leu Ala Tyr Ser Ile Thr Leu Val Thr Leu 450 455 460 Trp Ser Ile Trp His Tyr Ser 465 470 <210> 7 <211> 430 <212> PRT <213> artificial <220> <223> alpha7-GlyR chimeric receptor sequence <400> 7 Met Arg Cys Ser Pro Gly Gly Val Trp Leu Ala Leu Ala Ala Ser Leu 1 5 10 15 Leu His Val Ser Leu Gln Gly Glu Phe Gln Arg Lys Leu Tyr Lys Glu 20 25 30 Leu Val Lys Asn Tyr Asn Pro Leu Glu Arg Pro Val Ala Asn Asp Ser 35 40 45 Gln Pro Leu Thr Val Tyr Phe Ser Leu Ser Leu Leu Gln Ile Met Asp 50 55 60 Val Asp Glu Lys Asn Gln Val Leu Thr Thr Asn Ile Trp Leu Gln Met 65 70 75 80 Ser Trp Thr Asp His Tyr Leu Gln Trp Asn Val Ser Glu Tyr Pro Gly 85 90 95 Val Lys Thr Arg Phe Pro Asp Gly Gln Ile Trp Lys Pro Asp Ile Leu 100 105 110 Leu Tyr Asn Ser Ala Asp Glu Arg Phe Asp Ala Thr Phe His Thr Asn 115 120 125 Val Leu Val Asn Ser Ser Gly His Cys Gln Tyr Leu Pro Pro Gly Ile 130 135 140 Phe Lys Ser Ser Cys Tyr Ile Asp Val Arg Trp Phe Pro Phe Asp Val 145 150 155 160 Gln His Cys Lys Leu Lys Phe Gly Ser Trp Ser Tyr Gly Gly Trp Ser 165 170 175 Leu Asp Leu Gln Met Gln Glu Ala Asp Ile Ser Gly Tyr Ile Pro Asn 180 185 190 Gly Glu Trp Asp Leu Val Gly Ile Pro Gly Lys Arg Ser Glu Arg Phe 195 200 205 Tyr Glu Cys Cys Lys Glu Pro Tyr Pro Asp Val Thr Phe Thr Val Thr 210 215 220 Met Arg Arg Arg Met Gly Tyr Tyr Leu Ile Gln Met Tyr Ile Pro Ser 225 230 235 240 Leu Leu Ile Val Ile Leu Ser Trp Ile Ser Phe Trp Ile Asn Met Asp 245 250 255 Ala Ala Pro Ala Arg Val Gly Leu Gly Ile Thr Thr Val Leu Thr Met 260 265 270 Thr Thr Gln Ser Gly Ser Arg Ala Ser Leu Pro Lys Val Ser Tyr 275 280 285 Val Lys Ala Ile Asp Ile Trp Met Ala Val Cys Leu Leu Phe Val Phe 290,295,300 Ser Ala Leu Leu Glu Tyr Ala Ala Val Asn Phe Val Ser Arg Gln His 305 310 315 320 Lys Glu Leu Leu Arg Phe Arg Arg Lys Arg Arg His His Lys Glu Asp 325 330 335 Glu Ala Gly Glu Gly Arg Phe Asn Phe Ser Ala Tyr Gly Met Gly Pro 340 345 350 Cys Leu Gln Ala Lys Asp Gly Ile Ser Val Lys Gly Ala Asn Asn 355 360 365 Ser Asn Thr Thr Asn Pro Pro Pro Wing Pro Ser Lys Ser Pro Glu Glu 370 375 380 Met Arg Lys Leu Phe Ile Gln Arg Ala Lys Lys Ile Asp Lys Ile Ser 385 390 395 400 Arg Ile Gly Phe Pro Met Ala Phe Leu Ile Phe Asn Phe Tyr Trp 405 410 415 Ile Ile Tyr Lys Ile Val Arg Arg Glu Asp Val His Asn Gln 420 425 430 <210> 8 <211> 466 <212> PRT <213> artificial <220> <223> alpha7-5HT3 chimeric receptor sequence <400> 8 Met Arg Cys Ser Pro Gly Gly Val Trp Leu Ala Leu Ala Ala Ser Leu 1 5 10 15 Leu His Val Ser Leu Gln Gly Glu Phe Gln Arg Lys Leu Tyr Lys Glu 20 25 30 Leu Val Lys Asn Tyr Asn Pro Leu Glu Arg Pro Val Ala Asn Asp Ser 35 40 45 Gln Pro Leu Thr Val Tyr Phe Ser Leu Ser Leu Leu Gln Ile Met Asp 50 55 60 Val Asp Glu Lys Asn Gln Val Leu Thr Thr Asn Ile Trp Leu Gln Met 65 70 75 80 Ser Trp Thr Asp His Tyr Leu Gln Trp Asn Val Ser Glu Tyr Pro Gly 85 90 95 Val Lys Thr Val Arg Phe Pro Asp Gly Gln Ile Trp Lys Pro Asp Ile 100 105 110 Leu Leu Tyr Asn Ser Ala Asp Glu Arg Phe Asp Ala Thr Phe His Thr 115 120 125 Asn Val Leu Val Asn Ser Ser Gly His Cys Gln Tyr Leu Pro Pro Gly 130 135 140 Ile Phe Lys Ser Ser Cys Tyr Ile Asp Val Arg Trp Phe Pro Phe Asp 145 150 155 160 Val Gln His Cys Lys Leu Lys Phe Gly Ser Trp Ser Tyr Gly Gly Trp 165 170 175 Ser Leu Asp Leu Gln Met Gln Glu Ala Asp Ile Ser Gly Tyr Ile Pro 180 185 190 Asn Gly Glu Trp Asp Leu Val Gly Ile Pro Gly Lys Arg Ser Glu Arg 195 200 205 Phe Tyr Glu Cys Cys Lys Glu Pro Tyr Pro Asp Val Thr Phe Thr Val 210 215 220 Ile Ile Arg Arg Arg Pro Leu Phe Tyr Val Val Ser Leu Leu Leu Pro 225 230 235 240 Ser Ile Phe Leu Met Val Met Asp Ile Val Gly Phe Tyr Leu Pro Pro 245 250 255 Asn Ser Gly Glu Arg Val Ser Phe Lys Ile Thr Leu Leu Leu Gly Tyr 260 265 270 Ser Val Phe Leu Ile Ile Val Ser Asp Thr Leu Pro Ala Thr Ala Ile 275 280 285 Gly Thr Pro Leu Ile Gly Val Tyr Phe Val Val Cys Met Ala Leu Leu 290 295 300 Val Ile Ser Leu Ala Glu Thr Ile Phe Ile Val Arg Leu Val His Lys 305 310 315 320 Gln Asp Leu Gln Gln Pro Val Pro Ala Trp Leu Arg His Leu Val Leu 325 330 335 Glu Arg Ile Ala Trp Leu Leu Cys Leu Arg Glu Gln Ser Thr Ser Gln 340 345 350 Arg Pro Pro Ala Thr Ser Gln Ala Thr Lys Thr Asp Asp Cys Ser Ala 355 360 365 Met Gly Asn His Cys Ser His Met Gly Gly Pro Gln Asp Phe Glu Lys 370 375 380 Ser Pro Arg Asp Arg Cys Ser Pro Pro Pro Pro Pro Arg Glu Ala Ser 385 390 395 400 Leu Ala Val Cys Gly Leu Leu Gln Glu Leu Ser Ser Ile Arg Gln Phe 405 410 415 Leu Glu Lys Arg Asp Glu Ile Arg Glu Val Ala Arg Asp Trp Leu Arg 420 425 430 Val Gly Ser Val Leu Asp Lys Leu Leu Phe His Ile Tyr Leu Leu Ala 435 440 445 Val Leu Ala Tyr Ser Ile Thr Leu Val Met Leu Trp Ser Ile Trp Gln 450 455 460 Tyr Ala 465 <210> 9 <211> 195 <212> PRT <213> Humana <400> 9 Leu Leu Gln Thr Tyr Phe Pro Ala Thr Leu Met Val Met Leu Ser Trp 1 5 10 15 Val Ser Phe Trp Ile Asp Arg Arg Ala Val Pro Ala Arg Val Pro Leu 20 25 30 Gly Ile Thr Thr Val Leu Thr Met Ser Thr Ile Ile Thr Gly Val Asn 35 40 45 Ala Ser Met Pro Arg Val Ser Tyr Ile Lys Ala Val Asp Ile Tyr Leu 50 55 60 Trp Val Ser Phe Val Phe Val Phe Leu Ser Val Leu Glu Tyr Ala Ala 65 70 75 80 Val Asn Tyr Leu Thr Thr Val Gln Glu Arg Lys Glu Gln Lys Leu Arg 85 90 95 Glu Lys Leu Pro Cys Thr Ser Gly Leu Pro Pro Pro Arg Thr Ala Met 100 105 110 Leu Asp Gly Asn Tyr Ser Asp Gly Glu Val Asn Asp Leu Asp Asn Tyr 115 120 125 Met Pro Glu Asn Gly Glu Lys Pro Asp Arg Met Met Val Gln Leu Thr 130 135 140 Leu Ala Ser Glu Arg Ser Ser Pro Gln Arg Lys Ser Gln Arg Ser Ser 145 150 155 160 Tyr Val Ser Met Arg Ile Asp Thr His Ala Ile Asp Lys Tyr Ser Arg 165 170 175 Ile Ile Phe Pro Ala Ala Tyr Ile Leu Phe Asn Leu Ile Tyr Trp Ser 180 185 190 Ile Phe Ser 195 <210> 10 <211> 428 <212> PRT <213> artificial <220> <223> alpha7- GABAC chimeric receptor sequence <400> 10 Met Arg Cys Ser Pro Gly Gly Val Trp Leu Ala Leu Ala Ala Ser Leu 1 5 10 15 Leu His Val Ser Leu Gln Gly Glu Phe Gln Arg Lys Leu Tyr Lys Glu 20 25 30 Leu Val Lys Asn Tyr Asn Pro Leu Glu Arg Pro Val Ala Asn Asp Ser 35 40 45 Gln Pro Leu Thr Val Tyr Phe Ser Leu Ser Leu Leu Gln Ile Met Asp 50 55 60 Val Asp Glu Lys Asn Gln Val Leu Thr Thr Asn Ile Trp Leu Gln Met 65 70 75 80 Ser Trp Thr Asp His Tyr Leu Gln Trp Asn Val Ser Glu Tyr Pro Gly 85 90 95 Val Lys Thr Val Arg Phe Pro Asp Gly Gln Ile Trp Lys Pro Asp Ile 100 105 110 Leu Leu Tyr Asn Ser Ala Asp Glu Arg Phe Asp Ala Thr Phe His Thr 115 120 125 Asn Val Leu Val Asn Ser Ser Gly His Cys Gln Tyr Leu Pro Pro Gly 130 135 140 Ile Phe Lys Ser Ser Cys Tyr Ile Asp Val Arg Trp Phe Pro Phe Asp 145 150 155 160 Val Gln His Cys Lys Leu Lys Phe Gly Ser Trp Ser Tyr Gly Gly Trp 165 170 175 Ser Leu Asp Leu Gln Met Gln Glu Ala Asp Ile Ser Gly Tyr Ile Pro 180 185 190 Asn Gly Glu Trp Asp Leu Val Gly Ile Pro Gly Lys Arg Ser Glu Arg 195 200 205 Phe Tyr Glu Cys Cys Lys Glu Pro Tyr Pro Asp Val Thr Phe Thr Val 210 215 220 Thr Met Arg Arg Arg Thr Leu Tyr Tyr Leu Leu Gln Thr Tyr Phe Pro 225 230 235 240 Ala Thr Leu Met Val Met Leu Ser Trp Val Ser Phe Trp Ile Asp Arg 245 250 255 Arg Ala Val Pro Ala Arg Val Pro Leu Gly Ile Thr Thr Val Leu Thr 260 265 270 Met Ser Thr Ile Ile Thr Gly Val Asn Ala Ser Met Pro Arg Val Ser 275 280 285 Tyr Ile Lys Ala Val Asp Ile Tyr Leu Trp Val Ser Phe Val Phe Val 290 295 300 Phe Leu Ser Val Leu Glu Tyr Ala Ala Val Asn Tyr Leu Thr Thr Val 305 310 315 320 Gln Glu Arg Lys Glu Gln Lys Leu Arg Glu Lys Leu Pro Cys Thr Ser 325 330 335 Gly Leu Pro Pro Pro Arg Thr Ala Met Leu Asp Gly Asn Tyr Ser Asp 340 345 350 Gly Glu Val Asn Asp Leu Asp Asn Tyr Met Pro Glu Asn Gly Glu Lys 355 360 365 Pro Asp Arg Met Met Val Gln Leu Thr Leu Ala Ser Glu Arg Ser Ser 370 375 380 Pro Gln Arg Lys Ser Gln Arg Ser Ser Tyr Val Ser Met Arg Ile Asp 385 390 395 400 Thr His Ala Ile Asp Lys Tyr Ser Arg Ile Ile Phe Pro Ala Ala Tyr 405 410 415 Ile Leu Phe Asn Leu Ile Tyr Trp Ser Ile Phe Ser 420 425 <210> 11 <211> 233 <212> PRT <213> humane <400> 11 Met Arg Cys Ser Pro Gly Gly Val Trp Leu Ala Leu Ala Ala Ser Leu 1 5 10 15 Leu His Val Ser Leu Gln Gly Glu Phe Gln Arg Lys Leu Tyr Lys Glu 20 25 30 Leu Val Lys Asn Tyr Asn Pro Leu Glu Arg Pro Val Ala Asn Asp Ser 35 40 45 Gln Pro Leu Thr Val Tyr Phe Ser Leu Ser Leu Leu Gln Ile Met Asp 50 55 60 Val Asp Glu Lys Asn Gln Val Leu Thr Thr Asn Ile Trp Leu Gln Met 65 70 75 80 Ser Trp Thr Asp His Tyr Leu Gln Trp Asn Val Ser Glu Tyr Pro Gly 85 90 95 Val Lys Thr Val Arg Phe Pro Asp Gly Gln Ile Trp Lys Pro Asp Ile 100 105 110 Leu Leu Tyr Asn Ser Ala Asp Glu Arg Phe Asp Ala Thr Phe His Thr 115 120 125 Asn Val Leu Val Asn Ser Ser Gly His Cys Gln Tyr Leu Pro Pro Gly 130 135 140 Ile Phe Lys Ser Ser Cys Tyr Ile Asp Val Arg Trp Phe Pro Phe Asp 145 150 155 160 Val Gln His Cys Lys Leu Lys Phe Gly Ser Trp Ser Tyr Gly Gly Trp 165 170 175 Ser Leu Asp Leu Gln Met Gln Glu Ala Asp Ile Ser Gly Tyr Ile Pro 180 185 190 Asn Gly Glu Trp Asp Leu Val Gly Ile Pro Gly Lys Arg Ser Glu Arg 195 200 205 Phe Tyr Glu Cys Cys Lys Glu Pro Tyr Pro Asp Val Thr Phe Thr Val 210 215 220 Thr Met Arg Arg Arg Thr Leu Tyr Tyr 225 230 <210> 12 <211> 502 <212> PRT <213> rat <400> 12 Met Gly Gly Gly Arg Gly Gly Ile Trp Leu Ala Leu Ala Ala Ala Leu 1 5 10 15 Leu His Val Ser Leu Gln Gly Glu Phe Gln Arg Arg Leu Tyr Lys Glu 20 25 30 Leu Val Lys Asn Tyr Asn Pro Leu Glu Arg Pro Val Ala Asn Asp Ser 35 40 45 Gln Pro Leu Thr Val Tyr Phe Ser Leu Ser Leu Leu Gln Ile Met Asp 50 55 60 Val Asp Glu Lys Asn Gln Val Leu Thr Thr Asn Ile Trp Leu Gln Met 65 70 75 80 Ser Trp Thr Asp His Tyr Leu Gln Trp Asn Met Ser Glu Tyr Pro Gly 85 90 95 Val Lys Asn Val Arg Phe Pro Asp Gly Gln Ile Trp Lys Pro Asp Ile 100 105 110 Leu Leu Tyr Asn Ser Ala Asp Glu Arg Phe Asp Ala Thr Phe His Thr 115 120 125 Asn Val Leu Val Asn Ala Ser Gly His Cys Gln Tyr Leu Pro Pro Gly 130 135 140 Ile Phe Lys Ser Ser Cys Tyr Ile Asp Val Arg Trp Phe Pro Phe Asp 145 150 155 160 Val Gln Gln Cys Lys Leu Lys Phe Gly Ser Trp Ser Tyr Gly Gly Trp 165 170 175 Ser Leu Asp Leu Gln Met Gln Glu Ala Asp Ile Ser Ser Tyr Ile Pro 180 185 190 Asn Gly Glu Trp Asp Leu Met Gly Ile Pro Gly Lys Arg Asn Glu Lys 195 200 205 Phe Tyr Glu Cys Cys Lys Glu Pro Tyr Pro Asp Val Thr Tyr Thr Val 210 215 220 Thr Met Arg Arg Arg Thr Leu Tyr Tyr Gly Leu Asn Leu Leu Ile Pro 225 230 235 240 Cys Val Leu Ile Ser Ala Leu Ala Leu Leu Val Phe Leu Leu Pro Ala 245 250 255 Asp Ser Gly Glu Lys Ile Ser Leu Gly Ile Thr Val Leu Leu Ser Leu 260 265 270 Thr Val Phe Met Leu Leu Val Ala Glu Ile Met Pro Ala Thr Ser Asp 275 280 285 Ser Val Pro Leu Ile Ala Gln Tyr Phe Ala Ser Thr Met Ile Ile Val 290 295 300 Gly Leu Ser Val Val Val Thr Val Ile Val Leu Arg Tyr His His His 305 310 315 320 Asp Pro Asp Gly Gly Lys Met Pro Lys Trp Thr Arg Ile Ile Leu Leu 325 330 335 Asn Trp Cys Ala Trp Phe Leu Arg Met Lys Arg Pro Gly Glu Asp Lys 340 345 350 Val Arg Pro Ala Cys Gln His Lys Pro Arg Arg Cys Ser Leu Ala Ser 355 360 365 Val Glu Leu Ser Ala Gly Ala Gly Pro Pro Thr Ser Asn Gly Asn Leu 370 375 380 Leu Tyr Ile Gly Phe Arg Gly Leu Glu Gly Met His Cys Ala Pro Thr 385 390 395 400 Pro Asp Ser Gly Val Val Cys Gly Arg Leu Ala Cys Ser Pro Thr His 405 410 415 Asp Glu His Leu Met His Gly Ala His Pro Ser Asp Gly Asp Pro Asp 420 425 430 Leu Ala Lys Ile Leu Glu Glu Val Arg Tyr Ile Ala Asn Arg Asn Arg 435 440 445 Cys Gln Asp Glu Ser Glu Val Ile Cys Ser Glu Trp Lys Phe Ala Ala 450 455 460 Cys Val Val Asp Pro Leu Cys Leu Met Ala Phe Ser Val Phe Thr Ile 465 470 475 480 Ile Cys Thr Ile Gly Ile Leu Met Ser Ala Pro Asn Phe Val Glu Ala 485 490 495 Val Ser Lys Asp Phe Ala 500 <210> 13 <211> 438 <212> PRT <213> artificial <220> <223> PSAM4-GlyR-KirM3M4 sequence <400> 13 Met Arg Cys Ser Pro Gly Gly Val Trp Leu Ala Leu Ala Ala Ser Leu 1 5 10 15 Leu His Val Ser Leu Gln Gly Glu Phe Gln Arg Lys Leu Tyr Lys Glu 20 25 30 Leu Val Lys Asn Tyr Asn Pro Leu Glu Arg Pro Val Ala Asn Asp Ser 35 40 45 Gln Pro Leu Thr Val Tyr Phe Ser Leu Ser Leu Leu Gln Ile Met Asp 50 55 60 Val Asp Glu Lys Asn Gln Val Leu Thr Thr Asn Ile Trp Leu Gln Met 65 70 75 80 Ser Trp Thr Asp His Tyr Leu Gln Trp Asn Val Ser Glu Tyr Pro Gly 85 90 95 Val Lys Thr Val Arg Phe Pro Asp Gly Gln Ile Trp Lys Pro Asp Ile 100 105 110 Leu Leu Tyr Asn Ser Ala Asp Glu Arg Phe Asp Ala Thr Phe His Thr 115 120 125 Asn Val Gly Val Asn Ser Ser Gly His Cys Leu Tyr Leu Pro Pro Gly 130 135 140 Ile Phe Lys Ser Ser Cys Tyr Ile Asp Val Arg Trp Phe Pro Phe Asp 145 150 155 160 Val Gln His Cys Lys Leu Lys Phe Gly Ser Trp Ser Tyr Gly Gly Trp 165 170 175 Ser Leu Asp Leu Gln Met Gln Glu Ala Asp Ile Ser Gly Tyr Ile Pro 180 185 190 Asn Gly Glu Trp Asp Leu Val Gly Ile Pro Gly Lys Arg Ser Glu Arg 195 200 205 Phe Tyr Glu Cys Cys Lys Glu Pro Phe Pro Asp Val Thr Phe Thr Val 210 215 220 Thr Met Arg Arg Arg Met Gly Tyr Tyr Leu Ile Gln Met Tyr Ile Pro 225 230 235 240 Ser Leu Leu Ile Val Ile Leu Ser Trp Ile Ser Phe Trp Ile Asn Met 245 250 255 Asp Ala Ala Pro Ala Arg Val Gly Leu Gly Ile Thr Thr Val Leu Thr 260 265 270 Met Thr Thr Gln Ser Ser Gly Ser Arg Ala Ser Leu Pro Lys Val Ser 275 280 285 Tyr Val Lys Ala Ile Asp Ile Trp Met Ala Val Cys Leu Leu Phe Val 290 295 300 Phe Ser Ala Leu Leu Glu Tyr Ala Ala Val Asn Phe Val Ser Arg Gln 305 310 315 320 His Lys Glu Leu Leu Arg Phe Arg Arg Lys Arg Arg His His Lys Glu 325 330 335 Asp Glu Ala Gly Glu Gly Arg Phe Asn Phe Ser Ala Tyr Gly Met Gly 340 345 350 Pro Ala Cys Leu Gln Ala Lys Asp Gly Ile Ser Val Lys Gly Ala Asn 355 360 365 Asn Ser Asn Phe Cys Tyr Glu Asn Glu Val Thr Thr Asn Pro Pro Pro 370 375 380 Ala Pro Ser Lys Ser Pro Glu Glu Met Arg Lys Leu Phe Ile Gln Arg 385 390 395 400 Ala Lys Lys Ile Asp Lys Ile Ser Arg Ile Gly Phe Pro Met Ala Phe 405 410 415 Leu Ile Phe Asn Met Phe Tyr Trp Ile Ile Tyr Lys Ile Val Arg Arg 420 425 430 Glu Asp Val His Asn Gln 435 <210> 14 <211> 437 <212> PRT <213> artificial <220> <223> PSAM4-GlyR-B4sig sequence <400> 14 Met Arg Arg Ala Pro Ser Leu Val Leu Phe Phe Leu Val Ala Leu Cys 1 5 10 15 Gly Arg Gly Asn Cys Gly Glu Phe Gly Arg Lys Tyr Lys Glu Leu 20 25 30 Lys Asn Tyr Asn Pro Leu Glu Arg Pro Val Ala Asn Asp Ser Gln 35 40 45 Pro Leu Thr Val Tyr Phe Ser Leu Ser Leu Leu Gln Ile Met Asp Val 50 55 60 Asp Glu Lys Asn Gln Val Leu Thr Thr Asn Ile Trp Leu Gln Met Ser 65 70 75 80 Trp Thr Asp His Tyr Leu Gln Trp Asn Val Ser Glu Tyr Pro Gly Val 85 90 95 Lys Thr Val Arg Phe Pro Asp Gly Gln Ile Trp Lys Pro Asp Ile Leu 100 105 110 Leu Tyr Asn Ser Ala Asp Glu Arg Phe Asp Ala Thr Phe His Thr Asn 115 120 125 Val Gly Val Asn Ser Ser Gly His Cys Leu Tyr Leu Pro Pro Gly Ile 130 135 140 Phe Lys Ser Ser Cys Tyr Ile Asp Val Arg Trp Phe Pro Phe Asp Val 145 150 155 160 Gln His Cys Lys Leu Lys Phe Gly Ser Trp Ser Tyr Gly Gly Trp Ser 165 170 175 Leu Asp Leu Gln Met Gln Glu Ala Asp Ile Ser Gly Tyr Ile Pro Asn 180 185 190 Gly Glu Trp Asp Leu Val Gly Ile Pro Gly Lys Arg Ser Glu Arg Phe 195 200 205 Tyr Glu Cys Cys Lys Glu Pro Phe Pro Asp Val Thr Phe Thr Val Thr 210 215 220 Met Arg Arg Arg Met Gly Tyr Tyr Leu Ile Gln Met Tyr Ile Pro Ser 225 230 235 240 Leu Leu Ile Val Ile Leu Ser Trp Ile Ser Phe Trp Ile Asn Met Asp 245 250 255 Ala Ala Pro Ala Arg Val Gly Leu Gly Ile Thr Thr Val Leu Thr Met 260 265 270 Thr Thr Gln Ser Ser Gly Ser Arg Ala Ser Leu Pro Lys Val Ser Tyr 275 280 285 Val Lys Ala Ile Asp Ile Trp Met Ala Val Cys Leu Leu Phe Val Phe 290 295 300 Ser Ala Leu Leu Glu Tyr Ala Ala Val Asn Phe Val Ser Arg Gln His 305 310 315 320 Lys Glu Leu Leu Arg Phe Arg Arg Lys Arg Arg His His Lys Glu Asp 325 330 335 Glu Ala Gly Glu Gly Arg Phe Asn Phe Ser Ala Tyr Gly Met Gly Pro 340 345 350 Ala Cys Leu Gln Ala Lys Asp Gly Ile Ser Val Lys Gly Ala Asn Asn 355 360 365 Ser Asn Phe Cys Tyr Glu Asn Glu Val Thr Thr Asn Pro Pro Pro Ala 370 375 380 Pro Ser Lys Ser Pro Glu Glu Met Arg Lys Leu Phe Ile Gln Arg Ala 385 390 395 400 Lys Lys Ile Asp Lys Ile Ser Arg Ile Gly Phe Pro Met Ala Phe Leu 405 410 415 Ile Phe Asn Met Phe Tyr Trp Ile Ile Tyr Lys Ile Val Arg Arg Glu 420 425 430 Asp Val His Asn Gln 435 <210> 15 <211> 496 <212> PRT <213> artificial <220> <223> PSAM4-GlyR-Kv2M3M4-soma sequence <400> 15 Met Arg Cys Ser Pro Gly Gly Val Trp Leu Ala Leu Ala Ala Ser Leu 1 5 10 15 Leu His Val Ser Leu Gln Gly Glu Phe Gln Arg Lys Leu Tyr Lys Glu 20 25 30 Leu Val Lys Asn Tyr Asn Pro Leu Glu Arg Pro Val Ala Asn Asp Ser 35 40 45 Gln Pro Leu Thr Val Tyr Phe Ser Leu Ser Leu Leu Gln Ile Met Asp 50 55 60 Val Asp Glu Lys Asn Gln Val Leu Thr Thr Asn Ile Trp Leu Gln Met 65 70 75 80 Ser Trp Thr Asp His Tyr Leu Gln Trp Asn Val Ser Glu Tyr Pro Gly 85 90 95 Val Lys Thr Val Arg Phe Pro Asp Gly Gln Ile Trp Lys Pro Asp Ile 100 105 110 Leu Leu Tyr Asn Ser Ala Asp Glu Arg Phe Asp Ala Thr Phe His Thr 115 120 125 Asn Val Gly Val Asn Ser Ser Gly His Cys Leu Tyr Leu Pro Pro Gly 130 135 140 Ile Phe Lys Ser Ser Cys Tyr Ile Asp Val Arg Trp Phe Pro Phe Asp 145 150 155 160 Val Gln His Cys Lys Leu Lys Phe Gly Ser Trp Ser Tyr Gly Gly Trp 165 170 175 Ser Leu Asp Leu Gln Met Gln Glu Ala Asp Ile Ser Gly Tyr Ile Pro 180 185 190 Asn Gly Glu Trp Asp Leu Val Gly Ile Pro Gly Lys Arg Ser Glu Arg 195 200 205 Phe Tyr Glu Cys Cys Lys Glu Pro Phe Pro Asp Val Thr Phe Thr Val 210 215 220 Thr Met Arg Arg Arg Met Gly Tyr Tyr Leu Ile Gln Met Tyr Ile Pro 225 230 235 240 Ser Leu Leu Ile Val Ile Leu Ser Trp Ile Ser Phe Trp Ile Asn Met 245 250 255 Asp Ala Ala Pro Ala Arg Val Gly Leu Gly Ile Thr Thr Val Leu Thr 260 265 270 Met Thr Thr Gln Ser Ser Gly Ser Arg Ala Ser Leu Pro Lys Val Ser 275 280 285 Tyr Val Lys Ala Ile Asp Ile Trp Met Ala Val Cys Leu Leu Phe Val 290 295 300 Phe Ser Ala Leu Leu Glu Tyr Ala Ala Val Asn Phe Val Ser Arg Gln 305 310 315 320 His Lys Glu Leu Leu Arg Phe Arg Arg Lys Arg Arg His His Lys Glu 325 330 335 Asp Glu Ala Gly Glu Gly Arg Phe Asn Phe Ser Ala Tyr Gly Met Gly 340 345 350 Pro Ala Cys Leu Gln Ala Lys Asp Gly Ile Ser Val Lys Gly Ala Asn 355 360 365 Asn Ser Asn Gln Ser Gln Pro Ile Leu Asn Thr Lys Glu Met Ala Pro 370 375 380 Gln Ser Lys Pro Pro Glu Glu Leu Glu Met Ser Ser Met Pro Ser Pro 385 390 395 400 Val Ala Pro Leu Pro Ala Arg Thr Glu Gly Val Ile Asp Met Arg Ser 405 410 415 Met Ser Ser Ile Asp Ser Phe Ile Ser Cys Ala Thr Asp Phe Pro Glu 420 425 430 Ala Thr Arg Phe Thr Thr Asn Pro Pro Pro Ala Pro Ser Lys Ser Pro 435 440 445 Glu Glu Met Arg Lys Leu Phe Ile Gln Arg Ala Lys Lys Ile Asp Lys 450 455 460 Ile Ser Arg Ile Gly Phe Pro Met Ala Phe Leu Ile Phe Asn Met Phe 465 470 475 480 Tyr Trp Ile Ile Tyr Lys Ile Val Arg Arg Glu Asp Val His Asn Gln 485 490 495 <210> 16 <211> 7 <212> PRT <213> artificial <220> <223> endoplasmic reticulum export sequence <400> 16 Phe Cys Tyr Glu Asn Glu Val 1 5 <210> 17 <211> 21 <212> PRT <213> artificial <220> <223> CHRNB4 signal sequence <400> 17 Met Arg Arg Ala Pro Ser Leu Val Leu Phe Phe Leu Val Ala Leu Cys 1 5 10 15 Gly Arg Gly Asn Cys 20 <210> 18 <211> 65 <212> PRT <213> artificial <220> <223> KCNB1 somatic targeting sequence <400> 18 Gln Ser Gln Pro Ile Leu Asn Thr Lys Glu Met Ala Pro Gln Ser Lys 1 5 10 15 Pro Pro Glu Glu Leu Glu Met Ser Ser Met Pro Ser Pro Val Ala Pro 20 25 30 Leu Pro Ala Arg Thr Glu Gly Val Ile Asp Met Arg Ser Met Ser Ser 35 40 45 Ile Asp Ser Phe Ile Ser Cys Ala Thr Asp Phe Pro Glu Ala Thr Arg 50 55 60 Phe 65 <210> 19 <211> 1509 <212> DNA <213> rat <400> 19 atgggcggcg ggcgggggagg catctggctg gctctggccg cggcgctgct gcacgtgtcc 60 ctgcaaggcg agttccagag gaggctgtac aaggagctgg tcaagaacta caacccgctg 120 gagaggccgg tggccaacga ctcgcagccg ctcaccgtgt acttctccct gagtctcctg 180 cagatcatgg atgtggatga gaagaaccaa gttttaacca ccaacatttg gctacaaatg 240 tcttggacag atcactattt gcagtggaac atgtctgagt accccggagt gaagaatgtt 300 cgttttccag atggccagat ttggaaacca gacattctcc tctataacag tgctgatgag 360 cgctttgatg ccacgttcca caccaatgtt ttggtgaatg catctgggca ttgccagtat 420 ctccctccag gcatattcaa gagctcctgc tacattgacg ttcgctggtt cccttttgat 480 gtgcagcagt gcaaactgaa gtttgggtcc tggtcctatg gagggtggtc actggacctg 540 caaatgcaag aggcagatat cagcagctat atccccaacg gagaatggga tctcatggga 600 atccctggca aaaggaatga gaagttctat gagtgctgca aagagccata cccagatgtc 660 acctacacag taaccatgcg ccgtaggaca ctctactatg gcctcaatct gctcatccct 720 tgtgtactca tttcagccct ggctctgctg gtattcttgc tgcctgcaga ctctggagag 780 aaaatctctc ttggaataac tgtcttactt tctctgactg tcttcatgct gcttgtggct 840 gagatcatgc cagcaacatc tgattctgtg cccttgatag cacaatactt cgccagcacc 900 atgatcatcg tgggcctctc tgtagtggtg acagtgattg tgctgagata tcaccaccat 960 gaccctgatg gtggcaaaat gcctaagtgg accagaatca ttctcctgaa ctggtgtgca 1020 tggtttctgc gcatgaagag gcccggagag gacaaggtgc ggccagcttg tcagcacaag 1080 cctcggcgct gcagcctggc cagtgtggag ctgagtgcag gtgctgggcc acccaccagc 1140 aatggcaacc tgctctacat tggcttccga ggcctggagg gcatgcactg tgccccaact 1200 ccagactctg gggtcgtatg tggccgtttg gcctgctccc caacacatga tgagcacctc 1260 atgcacggtg cacacccctc tgatggggac cccgacctgg ccaagatcct ggaggaggtc 1320 cgctacatcg ccaaccgcaa ccgctgccag gacgagagtg aggtgatctg cagtgaatgg 1380 aagtttgcag cctgcgtggt ggacccgctt tgcctcatgg ccttttcggt ctttaccatc 1440 atctgtacca tcggcatcct catgtcagct ccaaactttg tggaggctgt gtccaaagac 1500 tttgcttaa 1509 <210> 20 <211> 672 <212> DNA <213> human <400> 20 atgcgctgtt ctccaggcgg cgtgtggctc gccctggctg cttcccttct gcacgttagc 60 ctgcagggtg agttccagcg caaactgtat aaggagcttg ttaagaatta taaccccctg 120 gagcggccgg tcgcaaatga ttcccagcca ctgacagtgt acttcagcct ctccttgctg 180 cagatcatgg acgtggatga aaaaccag gtgctgacca ctaatatttg gttgcagatg 240 tcctggaccg atcactactt gcagtggaat gtgagcgaat acccaggtgt aaagactgta 300 agattccctg acggccaaat ctggaaacca gatatcctgc tgtacaacag cgcagacgaa 360 aggtttgatg caacatttca caccaacgtg ttggtcaatt cttcaggcca ctgccagtac 420 ctgccccctg gaatcttcaa gtcctcatgc tatatcgacg tccgctggtt tcccttgac 480 gtccagcact gcaaactcaa attcgggagc tggagctacg gcggatggag cctggatctg 540 caaatgcagg aggctgacat ctctggttac atcccgaatg gggagtggga ccttgtggga 600 atccccggta aaagaagcga gcgattttat gaatgctgca aggaacccta ccctgacgta 660 acattcacag tt 672 <210> 21 <211> 687 <212> DNA <213> human <400> 21 atgcgctgtt ctccaggcgg cgtgtggctc gccctggctg cttcccttct gcacgttagc 60 ctgcagggtg agttccagcg caaactgtat aaggagcttg ttaagaatta taaccccctg 120 gagcggccgg tcgcaaatga ttcccagcca ctgacagtgt acttcagcct ctccttgctg 180 cagatcatgg acgtggatga aaaaccag gtgctgacca ctaatatttg gttgcagatg 240 tcctggaccg atcactactt gcagtggaat gtgagcgaat acccaggtgt aaagactgta 300 agattccctg acggccaaat ctggaaacca gatatcctgc tgtacaacag cgcagacgaa 360 aggtttgatg caacatttca caccaacgtg ttggtcaatt cttcaggcca ctgccagtac 420 ctgccccctg gaatcttcaa gtcctcatgc tatatcgacg tccgctggtt tcccttgac 480 gtccagcact gcaaactcaa attcgggagc tggagctacg gcggatggag cctggatctg 540 caaatgcagg aggctgacat ctctggttac atcccgaatg gggagtggga ccttgtggga 600 atccccggta aaagaagcga gcgattttat gaatgctgca aagagcccta cccagatgtc 660 accttcacag tgaccatgcg gagacgc 687 <210> 22 <211> 699 <212> DNA <213> human <400> 22 atgcgctgtt ctccaggcgg cgtgtggctc gccctggctg cttcccttct gcacgttagc 60 ctgcagggtg agttccagcg caaactgtat aaggagcttg ttaagaatta taaccccctg 120 gagcggccgg tcgcaaatga ttcccagcca ctgacagtgt acttcagcct ctccttgctg 180 cagatcatgg acgtggatga aaaaccag gtgctgacca ctaatatttg gttgcagatg 240 tcctggaccg atcactactt gcagtggaat gtgagcgaat acccaggtgt aaagactgta 300 agattccctg acggccaaat ctggaaacca gatatcctgc tgtacaacag cgcagacgaa 360 aggtttgatg caacatttca caccaacgtg ttggtcaatt cttcaggcca ctgccagtac 420 ctgccccctg gaatcttcaa gtcctcatgc tatatcgacg tccgctggtt tcccttcgac 480 gtccagcact gcaaactcaa attcgggagc tggagctacg gcggatggag cctggatctg 540 caaatgcagg aggctgacat ctctggttac atcccgaatg gggagtggga ccttgtggga 600 atccccggta aaagaagcga gcgattttat gaatgctgca aagagcccta cccagatgtc 660 accttcacag tgaccatgcg gagacgcaca ctgtattac 699 <210> 23 <211> 744 <212> DNA <213> mouse <400> 23 atcatcagaa gaaggccatt gttctacgcc gttagtttgt tgctccccag tatttttctc 60 atggtcgtgg acatcgtggg attttgtctc ccacctgata gcggggagag ggtctccttt 120 aagattacct tgttgctcgg ctattctgta tttctgatca tcgtgtccga tacccttcct 180 gccacaatcg gcactccgct gataggagtg tatttcgtcg tgtgtatggc actcctggtg 240 ataagtctgg cggaaactat cttcattgta cggctggtac ataagcagga cctgcaaaga 300 cccgtgccag actggttgcg acaccttgtg ctggacagaa ttgcatggat tctgtgtctt 360 ggcgagcaac ctatggccca ccggccacct gcaacctttc aagccaacaa gacagacgat 420 tgtagtgggt ctgatctgtt gcctgctatg gggaatcact gctcccatgt tgggggacca 480 caagatttgg aaaagacccc acgggggcgg ggatcacccc ttcctcctcc ccgagaagcc 540 tctctcgctg tccgggggct gctccaggaa ctgtcaagca tccgacattt tctggagaag 600 cgggacgaga tgagggaagt cgctagagac tggctgcgag tgggctacgt ccttgacagg 660 ctgctgtttc ggatctactt gctggcggtg ctggcttatt ccattactct ggtgacactc 720 tggtccatat ggcactacag ttag 744 <210> 24 <211> 726 <212> DNA <213> human <400> 24 atcatccgta gaaggcctct gttttacgtg gtgagcctgc tgctgccatc catcttcctg 60 atggtcatgg acatcgtggg cttttacctg ccacccaatt ctggcgagcg cgtgagcttc 120 aagatcacac tgctgctggg ctatagcgtg tttctgatca tcgtgtccga taccctgcct 180 gcaacagcaa tcggaacccc actgatcggc gtgtatttcg tggtgtgcat ggccctgctg 240 gtcatcagcc tggccgagac aatctttatc gtgcggctgg tgcacaagca ggacctgcag 300 cagcctgtgc cagcatggct gaggcacctg gtgctggaga ggatcgcatg gctgctgtgc 360 ctgagagagc agtccacatc tcagaggcct ccagccacct ctcaggccac caagacagac 420 gattgctctg ccatgggcaa tcactgtagc cacatgggcg gcccccagga ctttgagaag 480 tcccctcgcg atcggtgctc tccacctcca ccacctaggg aggccagcct ggccgtgtgc 540 ggcctgctgc aggagctgtc ctctatccgg cagttcctgg agaagcgcga cgagatccgg 600 gaggtggcca gagattggct gagggtgggc agcgtgctgg ataagctgct gtttcacatc 660 tacctgctgg cagtcctggc ctattctatt accctggtca tgctgtggtc catctggcag 720 tacgcc 726 <210> 25 <211> 609 <212> DNA <213> mouse <400> 25 atgggttatt atctgatcca aatgtatatc ccaagcttgc ttatagtgat tttgtcatgg 60 atctccttct ggattaatat ggacgccgct ccagctaggg tcggactggg catcaccaca 120 gtgctgacaa tgactactca gagctcaggc agccgagcca gcttgcccaa ggtttcttac 180 gtgaaggcca tcgatatctg gatggctgtc tgccttctgt ttgtcttcag cgcactgctg 240 gaatacgccg ctgtcaattt tgtgtctcga cagcataaag agctgttgcg gttcagaaga 300 aaacgacgcc accacaaaga ggatgaggca ggagaaggac gcttcaactt tagcgcctat 360 ggtatgggac ctgcttgcct ccaggctaaa gacggaattt ccgtgaaggg agccaacaat 420 agcaacacaa ccaacccacc ccctgctcca tctaagagcc cggaggaaat gcgcaaactc 480 tttattcaga gagcgaaaaa gatcgacaaa atctcccgga tcggattccc catggctttc 540 ctgattttca acatgtttta ttggatcatc tacaagattg tgcgaaggga ggacgtacac 600 aaccagtaa 609 <210> 26 <211> 588 <212> DNA <213> human <400> 26 cttttgcaaa cttactttcc agcaaccctc atggtgatgc tttcatgggt gtccttttgg 60 atcgaccgcc gagcggtccc tgcacgggtc cccctgggga ttacgacggt actgaccatg 120 agcaccataa tcactggagt caatgcaagc atgcctagag tgtcttacat aaaggccgtg 180 gacatctatc tgtgggttag ttttgtgttc gtattcctct ccgtgctgga gtatgcagct 240 gtgaactatc tgacaacagt tcaagagcgg aaagagcaga agttgaggga gaagctgcca 300 tgcactagcg gactgccacc gcccagaacc gctatgctcg atggtaacta ttccgacggc 360 gaagttaatg acctcgataa ctacatgcct gaaaatggcg aaaagcccga caggatgatg 420 gtccagctga cactggcctc agaaaggtcc agtccacaga gaaagtcaca gcgatcctct 480 tacgtcagca tgcgcatcga tacacatgcc atcgacaaat actctcgcat tatctttccg 540 gctgcttaca tattgttcaa ccttatctat tggagcattt tcagttga 588 <210> 27 <211> 1416 <212> DNA <213> artificial <220> <223> alpha7-5HT3 chimeric receptor sequence <400> 27 atgcgctgtt ctccaggcgg cgtgtggctc gccctggctg cttcccttct gcacgttagc 60 ctgcagggtg agttccagcg caaactgtat aaggagcttg ttaagaatta taaccccctg 120 gagcggccgg tcgcaaatga ttcccagcca ctgacagtgt acttcagcct ctccttgctg 180 cagatcatgg acgtggatga aaagaaccag gtgctgacca ctaatattg gttgcagatg 240 tcctggaccg atcactactt gcagtggaat gtgagcgaat acccaggtgt aaagactgta 300 agattccctg acggccaaat ctggaaacca gatatcctgc tgtacaacag cgcagacgaa 360 aggttgatg caacatttca caccaacgtg ttggtcaatt cttcaggcca ctgccagtac 420 ctgccccctg gaatcttcaa gtcctcatgc tatatcgacg tccgctggtt tcccttcgac 480 gtccagcact gcaaactcaa attcgggagc tggagctacg gcggatggag cctggatctg 540 caaatgcagg aggctgacat ctctggttac atcccgaatg gggagtggga ccttgtggga 600 atccccggta aaagaagcga gcgattttat gaatgctgca aggaacccta ccctgacgta 660 acattcacag ttatcatcag aagaaggcca ttgttctacg ccgttagttt gttgctcccc 720 agtatttttc tcatggtcgt ggacatcgtg ggattttgtc tcccacctga tagcggggag 780 agggtctcct ttaagattac cttgttgctc ggctattctg tatttctgat catcgtgtcc 840 gatacccttc ctgccacaat cggcactccg ctgataggag tgtatttcgt cgtgtgtatg 900 gcactcctgg tgataagtct ggcggaaact atcttcattg tacggctggt acataagcag 960 gacctgcaaa gacccgtgcc agactggttg cgacaccttg tgctggacag aattgcatgg 1020 attctgtgtc ttggcgagca acctatggcc caccggccac ctgcaacctt tcaagccaac 1080 aagacagacg attgtagtgg gtctgatctg ttgcctgcta tggggaatca ctgctcccat 1140 gttgggggac cacaagattt ggaaaagacc ccacgggggc ggggatcacc ccttcctcct 1200 ccccgagaag cctctctcgc tgtccggggg ctgctccagg aactgtcaag catccgacat 1260 tttctggaga agcgggacga gatgagggaa gtcgctagag actggctgcg agtgggctac 1320 gtccttgaca ggctgctgtt tcggatctac ttgctggcgg tgctggctta ttccattact 1380 ctggtgacac tctggtccat atggcactac agttag 1416 <210> 28 <211> 1296 <212> DNA <213> artificial <220> <223> alpha7-GlyR chimeric receptor sequence <400> 28 atgcgctgtt ctccaggcgg cgtgtggctc gccctggctg cttcccttct gcacgttagc 60 ctgcagggtg agttccagcg caaactgtat aaggagcttg ttaagaatta taaccccctg 120 gagcggccgg tcgcaaatga ttcccagcca ctgacagtgt acttcagcct ctccttgctg 180 cagatcatgg acgtggatga aaagaaccag gtgctgacca ctaatatttg gttgcagatg 240 tcctggaccg atcactactt gcagtggaat gtgagcgaat acccaggtgt aaagactgta 300 agattccctg acggccaaat ctggaaacca gatatcctgc tgtacaacag cgcagacgaa 360 aggtttgatg caacatttca caccaacgtg ttggtcaatt cttcaggcca ctgccagtac 420 ctgccccctg gaatcttcaa gtcctcatgc tatatcgacg tccgctggtt tcccttcgac 480 gtccagcact gcaaactcaa attcgggagc tggagctacg gcggatggag cctggatctg 540 caaatgcagg aggctgacat ctctggttac atcccgaatg gggagtggga ccttgtggga 600 atccccggta aaagaagcga gcgattttat gaatgctgca aagagcccta cccagatgtc 660 accttcacag tgaccatgcg gagacgcatg ggttattatc tgatccaaat gtatatccca 720 agcttgctta tagtgattt gtcatgatc tccttctgga ttatatgga cgccgctcca 780 gctagggtcg gactgggcat caccacagtg ctgacaatga ctactcagag ctcaggcagc 840 cgagccagct tgcccaggt ttcttacgtg aaggccatcg atatctggat ggctgtctgc 900 cttctgtttg tcttcagcgc actgctggaa tacgccgctg tcaatttgt gtctcgacag 960 cataaagagc tgttgcggtt cagaagaaa cgacgccacc aaagagga tgaggcagga 1020 gaaggacgct tcactttag cgcctatggt atgggacctg cttgcctcca ggctaagac 1080 ggaatttccg tgaagggagc siacaatagc aacaciacca acccacccc tgctccatct 1140 aagagcccgg aggaatgcg cieacttt attcagagag cgaaaagat cgacaaaatc 1200 tcccggatcg gattccccat ggctttcctg attttcaaca tgttttg gatcatctac 1260 aagattgtgc gagggagga cgtacaac spg 1296 <210> 29 <211> 1287 <212> DNA <213> artificial <220> <223> alpha7- GABAC chimeric receptor sequence <400> 29 atgcgctgtt ctccaggcgg cgtgtggctc gccctggctg cttcccttct gcacgttagc 60 ctgcagggtg agttccagcg caaactgtat aaggagcttg ttaagaatta taaccccctg 120 gagcggccgg tcgcaaatga ttcccagcca ctgacagtgt acttcagcct ctccttgctg 180 cagatcatgg acgtggatga aaagaaccag gtgctgacca ctaatatttg gttgcagatg 240 tcctggaccg atcactactt gcagtggaat gtgagcgaat acccaggtgt aaagactgta 300 agattccctg acggccaaat ctggaaacca gatatcctgc tgtacaacag cgcagacgaa 360 aggtttgatg caacatttca caccaacgtg ttggtcaatt cttcaggcca ctgccagtac 420 ctgccccctg gaatcttcaa gtcctcatgc tatatcgacg tccgctggtt tcccttcgac 480 gtccagcact gcaaactcaa attcgggagc tggagctacg gcggatggag cctggatctg 540 caaatgcagg aggctgacat ctctggttac atcccgaatg gggagtggga ccttgtggga 600 atccccggta aaagaagcga gcgattttat gaatgctgca aagagcccta cccagatgtc 660 accttcacag tgaccatgcg gagacgcaca ctgtattacc ttttgcaaac ttactttcca gcaaccctca tggtgatgct ttcatgggtg tccttttgga tcgaccgccg agcggtccct 780 gcacgggtcc ccctggggat tacgacggta ctgaccatga gcaccataat cactggagtc 840 aatgcaagca tgcctaggt gtcttacata aaggccgtgg acatctatct gtgggttagt tttgtgttcg tattcctctc cgtgctggag tatgcagctg tgaactatct gacaacagtt caagagcgga aagagcaga gttgagggag aagctgccat gcactagcgg actgccaccg cccagaaccg ctatgctcga tggtaactat tccgacggcg aagttaatga cctcgataac 1140. 1140. 1140. 1140. 1140. 1140. 1140. 1140. 1140. gaaaggtcca gtccacagag aaagtcacag cgatcctctt acgtcagcat gcgcatcgat 1260. acacatgcca tcgacaaata ctctcgcatt atctttccgg ctgcttacat attgttcaac cttatctatt ggagcatttt cagttga <210> 30 <211> 21 <212> DNA <213> artificial <220> <223> endoplasmic reticulum export sequence <400> 30 ttttgctatg aaaacgaagt c 21 <210> 31 <211> 63 <212> DNA <213> artificial <220> <223> CHRNB4 signal sequence <400> 31 atgagaaggg ccccatccct ggtattgttt tttttggtag ctttgtgcgg gagggggaac 60 tgc 63 <210> 32 <211> 195 <212> DNA <213> artificial <220> <223> KCNB1 somatic targeting sequence <400> 32 caaagccaac ctatccttaa cactaaagag agcgccgctc aatccaaacc caaagaagag 60 ttggaaatgg agtctatacc ttcacctgtt gcacctctcc ctactaggac cgaaggcgtg 120 attgacatgc gctctatgtc tagtatagat agctttatat cctgcgccac agactttccc 180 gaagccacta ggttc 195 <210> 33 <211> 7066 <212> DNA <213> artificial <220> <223> AAV-Syn::PSAM4-GlyR-IRES-EGFP-WPRE sequence <400> 33 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgcg 60 cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg 120 cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg 180 ttccttgtag ttaatgatta acccgccatg ctacttatct acgtagccat gctctaggaa 240 gatccgagct ccagtgtgct ggaattcgcc cttttcagta tttaaattga cgaccgaccc 300 cgacccactg gacaagcacc caacccccat tccccaaatt gcgcatcccc tatcagagag 360 ggggagggga aacaggatgc ggcgaggcgc gtgcgcactg ccagcttcag caccgcggac 420 agtgccttcg cccccgcctg gcggcgcgcg ccaccgccgc ctcagcactg aaggcgcgct 480 gacgtcactc gccggtcccc cgcaaactcc ccttcccggc caccttggtc gcgtccgcgc 540 cgccgccggc ccagccggac cgcaccacgc gaggcgcgag ataggggggc acgggcgcga 600 ccatctgcgc tgcggcgccg gcgactcagc gctgcctcag tctgcggtgg gcagcggagg 660 agtcgtgtcg tgcctgagag cgcagctgtg ctcctgggca ccgcgcagtc cgccccgcg 720 gctcctggcc agaccacccc taggaccccc tgccccaagt cgcagccgtt ggatcaggta 780 agtatcaagg ttacaagaca ggtttaagga gaccaataga aactgggctt gtcgagacag 840 agaagactct tgcgtttg ataggcacct attggtctta ctgacatcca ctttgccttt 900 ctctccacag gtgatgatat cactagtgct agcgccacca tgcgctgttc tccaggcggc 960 gtgtggctcg ccctggctgc ttcccttctg cacgttagcc tgcagggtga gttccagcgc 1020 aaactgtata aggagcttgt taagaattat aaccccctgg agcggccggt cgcaaatgat 1080 tcccagccac tgacagtgta cttcagcctc tccttgctgc agatcatgga cgtggatgaa 1140 aagaaccagg tgctgaccac tatatttgg ttgcagatgt cctggaccga tcactacttg 1200 cagtggaatg tgagcgaata cccaggtgta aagactgtaa gattccctga cggccaaatc 1260 tggaaaccag atacctgct gtacaacagc gcagacgaaa ggtttgatgc aacatttcac 1320 accaacgtgg gagtcaattc ttcaggccac tgcctgtacc tgccccctgg aatcttcaag 1380 tcctcatgct atacgacgt ccgctggttt cccttcgacg tccagcactg caaactcaaa 1440 ttcgggagct ggagctacgg cggatggagc ctggatctgc aaatgcagga ggctgacatc 1500 tctggttaca tcccgaatgg ggagtgggac cttgtgggaa tccccggtaa aagaagcgag 1560 cgattttatg aatgctgcaa agagcccttc ccagatgtca ccttcacagt gaccatgcgg 1620 agacgcatgg gttattatct gatccaaatg tatatcccaa gcttgcttat agtgattttg 1680 tcatggatct ccttctggat tatatggac gccgctccag ctagggtcgg actgggcatc 1740 accacagtgc tgacaatgac tactcagagc tcaggcagcc gagccagctt gcccaaggtt 1800 tcttacgtga aggccatcga tatctggatg gctgtctgcc ttctgtttgt cttcagcgca 1860 ctgctggaat acgccgctgt caatttgtg tctcgacagc ataaagagct gttgcggttc 1920 agaagaaaac gacgccacca caaagaggat gaggcaggag aaggacgctt caactttagc 1980 gcctatggta tgggacctgc ttgcctccag gctaaagacg gaatttccgt gaagggagcc 2040 aacaatagca acacaaccaa cccaccccct gctccatcta agagcccgga ggaaatgcgc 2100 aaactcttta ttcagagagc gaaaaagatc gacaaaatct cccggatcgg attccccatg 2160 gctttcctga ttttcaacat gttttattgg atcatctaca agattgtgcg aagggaggac 2220 gtacacaacc agtaagcggc cgcaattccc cccgcccccc cccccccccc ctcaccctcc 2280 ccccccccta acgttactgg ccgaagccgc ttggataag gccggtgtgc gtttgtctat 2340 atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggccccggaa acctggccct 2400 gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg 2460 2520 gcgacccttt gcaggcagcg gaaccccca cctggcgaca ggtgcctctg cggccaaaag 2580 ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg 2640 atagttgtgg aaagagtcaa atggctctcc taagcgtatt caacaagggg ctgaaggatg 2700 cccagaaggt accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat 2760 gtgtttagtc gaggtttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc 2820 tttgaaaaac acgatgataa tatggccaca accatgggag atccggtgag caagggcgag 2880 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac 2940 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag 3000 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc 3060 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag 3120 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac 3180 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg 3240 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac 3300 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc 3360 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac 3420 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc 3480 aagctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc 3540 gccgccggga tcactctcgg catggacgag ctgtacaagt aaaccggtaa tcaacctctg 3600 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta 3660 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt 3720 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc 3780 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt 3840 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg 3900 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac 3960 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc 4020 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac 4080 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct 4140 cagacgagtc ggatctccct ttgggccgcc tccccgccgc ttcgagcaga catgataaga 4200 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt 4260 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac 4320 aacaacaatt gcattcattt tatgtttcag gttcaggggg agatgtggga ggttttttaa 4380 agcaagtaaa acctctacaa atgtggtaaa atcgataagg atcttcctag agcatggcta 4440 cgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag tgatggagtt 4500 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg 4560 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct ggcgtaatag 4620 cgaagaggcc cgcaccgatc gccttccca acagttgcgc agcctgaatg gcgaatggga 4680 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 4740 tacacttgcc agcgccctag cgcccgctcc ttcgctttc ttcccttcct ttctcgccac 4800 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttaggt tccgatttag 4860 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc 4920 atcgccctga tagacggtttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 4980 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattct ttgatttata 5040 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa 5100 cgcgaatttt aaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg 5160 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 5220 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 5280 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 5340 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 5400 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 5460 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 5520 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 5580 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 5640 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 5700 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 5760 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 5820 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 5880 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 5940 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca 6000 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 6060 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 6120 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 6180 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 6240 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 6300 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 6360 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 6420 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag 6480 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 6540 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 6600 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 6660 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 6720 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 6780 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 6840 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 6900 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 6960 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 7020 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaag 7066 <210> 34 <211> 8167 <212> DNA <213> artificial <220> <223> AAV-CamkII::PSAM4-GlyR-IRES-EGFP-WPRE sequence <400> 34 cctgcaggca gctgcgcgct cgctcgctca ctgaggcgc ccgggcaaag cccggcgtc 60 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120 actccatcac taggggttcc tgcggccgca cgcgtttaac attatggcct taggtcactt 180 catctccatg gggttcttct tctgattttc tagaaaatga gatgggggtg cagagagctt 240 cctcagtgac ctgcccaggg tcacatcaga aatgtcagag ctagaacttg aactcagatt 300 actaatctta aattccatgc cttgggggca tgcaagtacg atatacagaa ggagtgaact 360 cattagggca gatgaccaat gagtttagga aagaagagtc cagggcaggg tacatctaca 420 ccacccgccc agccctgggt gagtccagcc acgttcacct cattatagtt gcctctctcc 480 agtcctacct tgacgggaag cacaagcaga aactgggaca ggagccccag gagaccaaat 540 cttcatggtc cctctgggag gatgggtggg gagagctgtg gcagaggcct caggaggggc 600 cctgctgctc agtggtgaca gataggggtg agaagcaga cagagtcatt ccgtcagcat 660 tctgggtctg tttggtactt cttctcacgc taaggtggcg gtgtgatatg cacaatggct 720 aaaaagcagg gagagctgga aaaaaaaagg gagagaga gaggccagt gagaacc 780 aattcccaga gghagg aaaccattac agagactaca aggggagg gaggagaga 840 tgaattagct tcccctgtaa accttagaac ccagctgttg ccagggcaac ggggcaatac 900 ctgtctcttc agaggagatg aagttgccag ggtaactaca tcctgtcttt ctcaggacc 960 atcccagaat gtggcacca ctagccgtta ccatagcaac tgcctcttg ccccacttaa 1020 tcccatcccg tctgttaaaa gggccctata gttggaggtg ggggaggtag gaagagcgat 1080 gatcacttgt gatcactagtt tgttcgcatc cccttctcca accccctcag tacatcaccc 1140 tggggaaca gggtccactt gctcctggggc ccacacagtc ctgcagtatt gtgtatataa 1200 ggccagggca agaggagca gttttaaag tgaaggcag gcaggtgttg gggaggcagt 1260 taccggggca acgggacag gggcgttcgg aggtggttgc catggggacc tggatgctga 1320 cgaaggctcg cgaggctgtg agcagccaca gtgccctgct cagaagcccc aagctcgtca 1380 gtcaagccgg ttctccgttt gcactcagga gcacgggcag gcgagtggcc cctagttctg 1440 ggggcagctc tagagcggta ccggatccag gtaagtatca aggttacaag acaggtttaa 1500 ggagaccaat agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca 1560 cctattggtc ttactgacat ccactttgcc tttctctcca caggtgatga attcgctagc 1620 gccaccatgc gctgttctcc aggcggcgtg tggctcgccc tggctgcttc ccttctgcac 1680 gttagcctgc agggtgagtt ccagcgcaaa ctgtataagg agcttgttaa gaattataac 1740 cccctggagc ggccggtcgc aaatgattcc cagccactga cagtgtactt cagcctctcc 1800 ttgctgcaga tcatggacgt ggatgaaaag aaccaggtgc tgaccactaa tatttggttg 1860 cagatgtcct ggaccgatca ctacttgcag tggaatgtga gcgaataccc aggtgtaaag 1920 actgtaagat tccctgacgg ccaaatctgg aaaccagata tcctgctgta caacagcgca 1980 gacgaaaggt ttgatgcaac atttcacacc aacgtgggag tcaattcttc aggccactgc 2040 ctgtacctgc cccctggaat cttcaagtcc tcatgctata tcgacgtccg ctggtttccc 2100 ttcgacgtcc agcactgcaa actcaaattc gggagctgga gctacggcgg atggagcctg 2160 gatctgcaaa tgcaggaggc tgacatctct ggttacatcc cgaatgggga gtgggacctt 2220 gtgggaatcc ccggtaaaag aagcgagcga ttttatgaat gctgcaaaga gcccttccca 2280 gatgtcacct tcacagtgac catgcggaga cgcatgggtt attatctgat ccaaatgtat 2340 atcccaagct tgcttatagt gattttgtca tggatctcct tctggattaa tatggacgcc 2400 gctccagcta gggtcggact gggcatcacc acagtgctga caatgactac tcagagctca 2460 ggcagccgag ccagcttgcc caaggtttct tacgtgaagg ccatcgatat ctggatggct 2520 gtctgccttc tgtttgtctt cagcgcactg ctggaatacg ccgctgtcaa ttttgtgtct 2580 cgacagcata aagagctgtt gcggttcaga agaaaacgac gccaccacaa agaggatgag 2640 gcaggagaag gacgcttcaa ctttagcgcc tatggtatgg gacctgcttg cctccaggct 2700 aaagacggaa tttccgtgaa gggagccaac aatagcaaca caaccaaccc accccctgct 2760 ccatctaaga gcccggagga aatgcgcaaa ctctttattc agagcgaa aaagatcgac 2820 aaaatctccc ggatcggatt ccccatggct ttcctgattt tcaacatgtt ttattggatc 2880 atctacaaga ttgtgcgaag ggaggacgta caaaccagt aagcggccgc aattcccccc 2940 gcccccccccc cccccccctc accctccccc cccctaacg ttactggccg aagccgcttg 3000 gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc 3060 aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc 3120 cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa 3180 gcttcttgaa ggaaacac gtctgtagcg accctttgca ggcagcgggaa ccccccacct 3240 ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca 3300 caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctaa 3360 gcgtattcaa caaggggctg areatgccc agaaggtacc ccattgtatg ggatctgatc 3420 tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc 3480 ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatat ggccacaacc 3540 atgggagatc cggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc 3600 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat 3660 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc 3720 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac 3780 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc 3840 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc 3900 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc 3960 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag 4020 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg 4080 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc 4140 gacaaccact acctgagcac ccagtccaag ctgagcaaag accccaacga gaagcgcgat 4200 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg 4260 tacaagtaaa ccggtgtcga caagcttatc gataatcaac ctctggatta caaaatttgt 4320 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct 4380 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat 4440 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg 4500 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag 4560 ctcctttccg gaactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc 4620 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg 4680 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc 4740 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc 4800 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc 4860 tccctttggg ccgcctcccc gcatcgatac cgagcgctgc tcgagagatc tacgggtggc 4920 atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact ccagtgccca 4980 ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg tccttctata 5040 atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag acaacctgta 5100 gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct tggctcactg 5160 caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag ttgttgggat 5220 tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga cggggtttca 5280 ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca ccttggcctc 5340 ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt ctgattttgt 5400 aggtaaccac gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc 5460 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg 5520 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat 5580 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca 5640 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 5700 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 5760 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 5820 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt 5880 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 5940 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat 6000 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa 6060 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca 6120 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg 6180 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg 6240 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc 6300 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt 6360 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 6420 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 6480 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 6540 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 6600 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 6660 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 6720 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 6780 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 6840 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 6900 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 6960 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 7020 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 7080 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 7140 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 7200 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 7260 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 7320 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 7380 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 7440 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 7500 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 7560 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 7620 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg 7680 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 7740 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 7800 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 7860 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 7920 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 7980 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 8040 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 8100 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 8160 cacatgt 8167

Claims

1. 1. A modified ligand-gated ion channel (LGIC) comprising at least one modified LGIC subunit, the modified LGIC subunit comprising: a ligand binding domain (LBD) comprising an amino acid modification; an ionic pore domain (IPD); 1. A modified ligand-gated ion channel comprising:

2. The modified LGIC of claim 1, which is a chimeric LGIC comprising an LBD from a first LGIC and an IPD from a second LGIC.

3. The modified LGIC of claim 1 , wherein the LBD is an alpha 7 nicotinic acetylcholine receptor (α7-nAChR) LBD.

4. 4. The modified LGIC of claim 3, wherein the amino acid modification comprises an amino acid substitution at one or more amino acid residues selected from the group consisting of residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, and 219 of the α7-nAChR LBD.

5. 5. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 77 of the α7-nAChR LBD, and the amino acid substitution is selected from the group consisting of W77F and W77Y.

6. 5. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 79 of the α7-nAChR LBD, and the amino acid substitution is selected from the group consisting of Q79A, Q79G, and Q79S.

7. 5. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 115 of the α7-nAChR LBD, and the amino acid substitution is a Y115F substitution.

8. 5. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 131 of the α7-nAChR LBD, and the amino acid substitution is selected from the group consisting of L131A, L131G, L131M, and L131N.

9. 5. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 139 of the α7-nAChR LBD, and the amino acid substitution is selected from the group consisting of Q139G and Q139L.

10. 5. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 175 of the α7-nAChR LBD and is selected from the group consisting of G175A, G175F, G175H, G175K, G175M, G175R, G175S, and G175V.

11. 5. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 210 of the α7-nAChR LBD, and the amino acid substitution is a Y210F substitution.

12. 5. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 216 of the α7-nAChR LBD, and the amino acid substitution is a P216I substitution.

13. 5. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 217 of the α7-nAChR LBD, and the amino acid substitution is a Y217F substitution.

14. 5. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 219 of the α7-nAChR LBD, and the amino acid substitution is a D219A substitution.

15. The modified LGIC of claim 4, wherein the α7-nAChR LBD comprises an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution.

16. The modified LGIC of claim 4, wherein the α7-nAChR LBD comprises an L131M amino acid substitution and a Y115F amino acid substitution.

17. The modified LGIC of claim 4, wherein the α7-nAChR LBD comprises a W77F amino acid substitution, a Q79G amino acid substitution, and a G175K amino acid substitution.

18. The modified LGIC of claim 4, wherein the α7-nAChR LBD comprises a Q79G amino acid substitution, a Y115F amino acid substitution, and a G175K amino acid substitution.

19. The modified LGIC of claim 4, wherein the α7-nAChR LBD comprises a Y115F amino acid substitution and a G175K amino acid substitution.

20. The modified LGIC of claim 4, wherein the α7-nAChR LBD comprises a Q79G amino acid substitution and a 216I amino acid substitution.

21. 2. The modified LGIC of claim 1, wherein the IPD is from a receptor selected from the group consisting of a serotonin 3 receptor (5HT3) IPD, a glycine receptor (GlyR) IPD, a gamma-aminobutyric acid (GABA) receptor IPD, and an alpha 7 nicotinic acetylcholine receptor (α7-nAChR) IPD.

22. 22. The modified LGIC of claim 21, wherein the IPD comprises an amino acid substitution at residue 298.

23. 23. The modified LGIC of claim 22, wherein the IPD is a GlyR IPD and the amino acid substitution is an A298G substitution.

24. 23. The modified LGIC of claim 22, wherein the IPD is a GABA IPD and the amino acid substitution is a W298A substitution.

25. The modified LGIC of claim 1, wherein an exogenous LGIC ligand activates the modified LGIC, and the exogenous LGIC ligand is a synthetic exogenous LGIC ligand selected from the group consisting of quinuclidine, tropane, 9-azabicyclo[3.3.1]nonane, 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, and 1,4-diazabicyclo[3.2.2]nonane.

26. 26. The modified LGIC of claim 25, wherein the synthetic exogenous LGIC ligand is a tropane, and the tropane is selected from the group consisting of tropisetron, pseudotropisetron, nortropisetron, compound 723, compound 725, compound 737, and compound 745.

27. 26. The modified LGIC of claim 25, wherein the synthetic exogenous LGIC ligand is a quinuclidine selected from the group consisting of PNU-282987, PHA-543613, Compound 0456, Compound 0434, Compound 0436, Compound 0354, Compound 0353, Compound 0295, Compound 0296, Compound 0536, Compound 0676, and Compound 702.

28. 26. The modified LGIC of claim 25, wherein the synthetic exogenous LGIC ligand is 9-azabicyclo[3.3.1]nonane, and the 9-azabicyclo[3.3.1]nonane is compound 536.

29. 26. The modified LGIC of claim 25, wherein the synthetic exogenous LGIC ligand is a 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, and the 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine is selected from the group consisting of varenicline, compound 765, and compound 770.

30. 26. The modified LGIC of claim 25, wherein the synthetic exogenous LGIC ligand is 1,4-diazabicyclo[3.2.2]nonane, and the 1,4-diazabicyclo[3.2.2]nonane is selected from the group consisting of 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-dioxide, Compound 773, and Compound 774.

31. 2. The modified LGIC of claim 1, wherein the LBD is an α7-nAChR LBD and the α7-nAChR LBD further comprises at least one modified amino acid that confers selective binding to another α7-nAChR LBD having at least one modified amino acid over binding to an unmodified LGIC.

32. The modified LGIC of claim 31 , wherein the unmodified LGIC is an endogenous LGIC.

33. The modified LGIC of claim 32, wherein the endogenous LGIC is an endogenous α7-nAChR.

34. 32. The modified LGIC of claim 31, wherein the at least one modified amino acid that confers selective binding comprises an amino acid substitution at amino acid residue 27 and / or residue 41 of the α7-nAChR LBD.

35. 35. The modified LGIC of claim 34, wherein the at least one modified amino acid comprises an R27D substitution and / or an E41R substitution.

36. The modified LGIC of claim 1, wherein the IPD is a mouse 5HT3 IPD, and the mouse 5HT3 IPD further comprises at least one modified amino acid that confers increased ionic conductivity to the modified LGIC.

37. The modified LGIC of claim 36, wherein at least one modified amino acid in mouse 5HT3 IPD that confers increased ionic conductivity to the modified LGIC comprises an amino acid substitution at amino acid residues 425, 429, and / or 433 of mouse 5HT3 IPD.

38. 38. The modified LGIC of claim 37, wherein at least one modified amino acid comprises an R425Q substitution, an R429D substitution, and / or an R433A substitution.

39. The modified LGIC of claim 1, wherein the IPD is a human 5HT3 IPD, and the human 5HT3 IPD further comprises at least one modified amino acid that confers increased ionic conductivity to the modified LGIC.

40. The modified LGIC of claim 39, wherein at least one modified amino acid in human 5HT3 IPD that confers increased ionic conductivity to the modified LGIC comprises an amino acid substitution at amino acid residues 420, 424, and / or 428 of human 5HT3 IPD.

41. 41. The modified LGIC of claim 40, wherein at least one modified amino acid comprises an R420Q substitution, an R424D substitution, and / or an R428A substitution.

42. The modified LGIC of claim 1 , wherein the LBD has reduced binding to an endogenous LGIC ligand.

43. 43. The modified LGIC of claim 42, wherein the endogenous LGIC ligand is acetylcholine (ACh).

44. 44. The modified LGIC of claim 43, wherein the modified LGIC has an EC50 for ACh of greater than 20 μM.

45. 1. A ligand with increased potency for modified ligand-gated ion channels (LGICs), comprising the compound of formula I: (In the formula, Each of X1, X2, and X3 is independently CH, CH2, O, NH, or NMe; each n is independently 0 or 1; Y is O or S; A is an aromatic substituent; R is H or pyridinylmethylene. A ligand comprising:

46. 46. ​​The ligand of claim 45, wherein the aromatic substituent is selected from the group consisting of 1H-indole, 4-(trifluoromethyl)benzene, 2,5-dimethoxybenzene, 4-chloroaniline, aniline, 5-(trifluoromethyl)pyridin-2-yl, 6-(trifluoromethyl)nicotine, and 4-chloro-benzene.

47. Formula II: (In the formula, X3 is O, NH, or CH2; Y is O or S; A is an aromatic substituent; R is H or pyridinylmethylene.

46. ​​The ligand of claim 45, which is a quinuclidine having the formula:

48. 48. The ligand of claim 47, wherein the aromatic substituent is selected from the group consisting of 1H-indole, 4-(trifluoromethyl)benzene, 4-chlorobenzene, 2,5-dimethoxybenzene, 4-(trifluoromethyl)benzene, 4-chloroaniline, aniline, 5-(trifluoromethyl)pyridin-2-yl, 6-(trifluoromethyl)nicotine, 3-chloro-4-fluorobenzene, and 1H-indole.

49. 48. The ligand of claim 47, wherein the quinuclidine is selected from the group consisting of PNU-282987, PHA-543613, Compound 0456, Compound 0434, Compound 0436, Compound 0354, Compound 0353, Compound 0295, Compound 0296, Compound 0536, Compound 0676, and Compound 0702.

50. Formula III: (In the formula, X2 is NH or NMe; X3 is O, NH, or CH2; Y is O or S; A is an aromatic substituent.

46. ​​The ligand of claim 45, which is a tropane having the formula:

51. 51. The ligand of claim 50, wherein said aromatic substituent is selected from the group consisting of 1H-indole, 1H-indazole, 7-methoxy-1H-indole, 7-methyl-1H-indole, and 5-chloro-1H-indole.

52. 51. The ligand of claim 50, wherein the tropane is selected from the group consisting of tropisetron, pseudotropisetron, nortropisetron, compound 723, compound 725, compound 737, and compound 745.

53. Formula IV: (In the formula, X2 is NH or NMe; X3 is O, NH, or CH; Y is O or S; A is an aromatic substituent.

46. ​​The ligand of claim 45, which is a 9-azabicyclo[3.3.1]nonane having the formula:

54. 55. The ligand of claim 54, wherein said aromatic substituent is selected from the group consisting of 4-chloro-benzene, 1H-indole, 1H-indazole, 7-methoxy-1H-indazole.

55. 55. The ligand of claim 54, wherein said 9-azabicyclo[3.3.1]nonane is selected from the group consisting of compound 0536, compound 0749, compound 0751, compound 0760, and compound 0763.

56. 1. A ligand with increased potency for modified ligand-gated ion channels (LGICs), comprising the compound of formula V: (In the formula, R1 is H, phenyl, 2-tolyl, 3-pyridyl, 4-pyridyl, trifluoromethyl, methoxy, ethoxy, propoxy, isopropoxy, N,N-dimethylamino, N,N-diethylamino, imidazole, pyrrole, pyrazole, triazole, or isoxazol-3-amine; R2 is H, methyl, or phenyl. A ligand comprising:

57. 57. The ligand of claim 56, wherein the 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine is selected from the group consisting of Compound 0765, Compound 0770, Compound 0780, Compound 0782, Compound 0785, Compound 0788, Compound 0782, Compound 0789, Compound 0791, Compound 0793, Compound 0794, Compound 0795, Compound 0798, Compound 0799, Compound 0800, Compound 0801, Compound 0802, Compound 0803, Compound 0804, Compound 0805, Compound 0807, Compound 0808, Compound 0812, Compound 0813, Compound 815, Compound 816, and Compound 817.

58. 1. A ligand with increased potency for modified ligand-gated ion channels (LGICs), comprising the compound of formula VII: (wherein R is H, F, or NO) ２ is) A ligand comprising:

59. 59. The ligand of claim 58, wherein the 1,4-diazabicyclo[3.2.2]nonane is selected from the group consisting of 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-dioxide, compound 0773, and compound 0774.

60. 1. A method of treating a channelopathy in a mammal, comprising: administering to cells in the mammal a modified ligand-gated ion channel (LGIC), wherein an exogenous LGIC ligand selectively binds to the modified LGIC, the modified LGIC comprising at least one modified LGIC subunit, the modified LGIC subunit comprising: a ligand-binding domain comprising at least one modified amino acid; and Ionic Pore Domain and administering the exogenous ligand to a mammal; A method comprising:

61. 61. The method of claim 60, wherein the channelopathy is selected from the group consisting of Bartter syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT), congenital hyperinsulinemia, cystic fibrosis, Dravet syndrome, transient ataxia, erythromelalgia, generalized epilepsy (e.g., with febrile seizures), familial hemiplegic migraine, fibromyalgia, hyperkalemic periodic paralysis, hypokalemic periodic paralysis, Lambert-Eaton myasthenic syndrome, long QT syndrome (e.g., Romano-Ward syndrome), short QT syndrome, malignant hyperthermia, mucolipidosis type IV, myasthenia gravis, myotonia congenita, neuromyelitis optica, neuromyotonia, nonsyndromic hearing loss, congenital paramyotonia, retinitis pigmentosa, Timothy syndrome, tinnitus, seizures, trigeminal neuralgia, and multiple sclerosis.

62. 1. A method for modulating ion transport across a cell membrane of a mammalian cell, comprising: administering to a cell a modified ligand-gated ion channel (LGIC), wherein the exogenous LGIC ligand selectively binds to the modified LGIC, the modified LGIC comprising at least one modified LGIC subunit, the modified LGIC subunit comprising: a ligand-binding domain comprising at least one modified amino acid; and Ionic Pore Domain and administering the exogenous ligand to a mammal; A method comprising:

63. 63. The method of claim 62, wherein the modulation comprises activating ion transport.

64. 63. The method of claim 62, wherein said modulation comprises inhibiting ion transport.

65. 63. The method of claim 62, wherein the cell is selected from the group consisting of a neuron, a glial cell, a muscle cell, a stem cell, an endocrine cell, and an immune cell.

66. 63. The method of claim 62, wherein administering the modified LGIC to a cell comprises in vivo administration.

67. 63. The method of claim 62, wherein administering the modified LGIC to a cell comprises ex vivo administration.

68. 1. A method of modulating cellular excitability in a mammal, comprising: administering to the cell a modified ligand-gated ion channel (LGIC), wherein the exogenous LGIC ligand selectively binds to the modified LGIC, the modified LGIC comprising at least one modified LGIC subunit, the modified LGIC subunit comprising: a ligand-binding domain comprising at least one modified amino acid; and Ionic Pore Domain and administering the exogenous ligand to a mammal; A method comprising:

69. 69. The method of claim 68, wherein the modulation comprises increasing the excitability of the cell.

70. 69. The method of claim 68, wherein the modulation comprises decreasing cellular excitability.

71. 69. The method of claim 68, wherein the cell is an excitable cell.

72. 69. The method of claim 68, wherein the cell is selected from the group consisting of a neuron, a glial cell, a muscle cell, a stem cell, an endocrine cell, and an immune cell.

73. 69. The method of claim 68, wherein administering the modified LGIC to a cell comprises in vivo administration.

74. 69. The method of claim 68, wherein administering the modified LGIC to a cell comprises ex vivo administration.

75. 1. A method of modulating cellular activity in a mammal, comprising: administering to the cell a modified ligand-gated ion channel (LGIC), wherein the exogenous LGIC ligand selectively binds to the modified LGIC, the modified LGIC comprising at least one modified LGIC subunit, the modified LGIC subunit comprising: a ligand-binding domain comprising at least one modified amino acid; and Ionic Pore Domain and administering the exogenous ligand to a mammal; A method comprising:

76. 76. The method of claim 75, wherein the modulation comprises increasing an activity of the cell.

77. 76. The method of claim 75, wherein the modulation comprises reducing an activity of the cell.

78. 76. The method of claim 75, wherein the activity is selected from the group consisting of ion transport, passive transport, excitation, inhibition, and exocytosis.

79. 76. The method of claim 75, wherein the cell is selected from the group consisting of a neuron, a glial cell, a muscle cell, a stem cell, an endocrine cell, and an immune cell.

80. 76. The method of claim 75, wherein administering the modified LGIC to a mammalian cell comprises in vivo administration.

81. 76. The method of claim 75, wherein administering the modified LGIC to a mammalian cell comprises ex vivo administration.

82. 76. The modified LGIC of any one of claims 60, 62, 68, or 75, wherein the modified LGIC is a chimeric LGIC comprising an LBD from a first LGIC and an IPD from a second LGIC.

83. 83. The method of claim 82, wherein the chimeric LGIC is a homomeric chimeric LGIC.

84. 76. The method of any one of claims 60, 62, 68, or 75, wherein administering the modified LGIC to a cell comprises administering a nucleic acid encoding the modified LGIC.

85. 85. The method of claim 84, wherein the modified LGIC comprises a sequence having at least 85% identity to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:

10.

86. 86. The method of claim 85, wherein the modified LGIC comprises a sequence having at least 90% identity to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:

10.

87. 87. The method of claim 86, wherein the modified LGIC comprises a sequence having at least 95% identity to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO:

10.

88. 88. The method of claim 87, wherein the modified LGIC comprises the sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:

10.

89. 76. The method of any one of claims 60, 62, 68, or 75, wherein the LBD is an alpha 7 nicotinic acetylcholine receptor (α7-nAChR) LBD.

90. 90. The method of claim 89, wherein the at least one modified amino acid in the α7-nAChR LBD comprises an amino acid substitution at at least one amino acid residue selected from the group consisting of residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, and 219 of the α7-nAChR LBD.

91. 91. The method of claim 90, wherein the amino acid substitution is at residue 77 of the α7-nAChR LBD, and the amino acid substitution is selected from the group consisting of W77F and W77Y.

92. 91. The method of claim 90, wherein the amino acid substitution is at residue 79 of the α7-nAChR LBD, and the amino acid substitution is selected from the group consisting of Q79A, Q79G, and Q79S.

93. 91. The method of claim 90, wherein the amino acid substitution is at residue 115 of the α7-nAChR LBD, and the amino acid substitution is a Y115F substitution.

94. 91. The method of claim 90, wherein the amino acid substitution is at residue 131 of the α7-nAChR LBD, and the amino acid substitution is selected from the group consisting of L131A, L131G, L131M, and L131N.

95. 91. The method of claim 90, wherein the amino acid substitution is at residue 139 of the α7-nAChR LBD, and the amino acid substitution is a Q139G or Q139L substitution.

96. 91. The method of claim 90, wherein the amino acid substitution is at residue 175 of the α7-nAChR LBD, and the amino acid substitution is selected from the group consisting of G175A, G175F, G175H, G175K, G175M, G175R, G175S, and G175V.

97. 91. The method of claim 90, wherein the amino acid substitution is at residue 210 of the α7-nAChR LBD, and the amino acid substitution is a Y210F substitution.

98. 91. The method of claim 90, wherein the amino acid substitution is at residue 216 of the α7-nAChR LBD, and the amino acid substitution is P216I.

99. 91. The method of claim 90, wherein the amino acid substitution is at residue 217 of the α7-nAChR LBD, and the amino acid substitution is Y217F.

100. 91. The method of claim 90, wherein the amino acid substitution is at residue 219 of the α7-nAChR LBD, and the amino acid substitution is D219A.

101. 76. The method of any one of claims 60, 62, 68, or 75, wherein the IPD comprises at least one modified amino acid.

102. 102. The method of claim 101, wherein the IPD is selected from the group consisting of a serotonin 3 receptor (5HT3) IPD, a glycine receptor (GlyR) IPD, a GABA receptor IPD, and an alpha 7 nicotinic acetylcholine receptor (α7-nAChR) IPD.

103. 103. The method of claim 102, wherein the IPD is a GlyR IPD and the at least one modified amino acid comprises an amino acid substitution at residue 298 of modified LGIC.

104. 104. The method of claim 103, wherein the amino acid substitution at residue 298 of the modified LGIC is an A298G substitution.

105. 103. The modified LGIC of claim 102, wherein the IPD is a GABA IPD and the at least one modified amino acid in the GABA IPD comprises an amino acid substitution at residue 298 of the modified LGIC.

106. The modified LGIC of claim 105, wherein the amino acid substitution at residue 298 of the chimeric LGIC is a W298A substitution.

107. The modified LGIC of any one of claims 60, 62, 68, or 75, wherein the exogenous LGIC ligand is a synthetic exogenous LGIC ligand.

108. 108. The method of claim 107, wherein the synthetic exogenous LGIC ligand is selected from the group consisting of tropane, quinuclidine, 9-azabicyclo[3.3.1]nonane, 1,4-diazabicyclo[3.2.2]nonane, and 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine.

109. The method of claim 108, wherein the synthetic exogenous LGIC ligand is a tropane, and the tropane is selected from the group consisting of tropisetron, pseudotropisetron, nortropisetron, compound 723, compound 725, compound 737, and compound 745.

110. 109. The method of claim 108, wherein the synthetic exogenous LGIC ligand is quinuclidine, and the quinuclidine is selected from the group consisting of PNU-282987, PHA-543613, Compound 0456, Compound 0434, Compound 0436, Compound 0354, Compound 0353, Compound 0295, Compound 0296, Compound 0536, Compound 0676, and Compound 702.

111. 109. The method of claim 108, wherein the synthetic exogenous LGIC ligand is 9-azabicyclo[3.3.1]nonane, and the 9-azabicyclo[3.3.1]nonane is selected from the group consisting of Compound 0536, Compound 0749, Compound 0751, Compound 0760, and Compound 0763.

112. 109. The method of claim 108, wherein the synthetic exogenous LGIC ligand is 1,4-diazabicyclo[3.2.2]nonane, and the 1,4-diazabicyclo[3.2.2]nonane is selected from the group consisting of 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-dioxide, compound 0773, and compound 0774.

113. 109. The method of claim 108, wherein the synthetic exogenous LGIC ligand is 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, and the 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine is selected from the group consisting of varenicline, compound 0765, and compound 0770.

114. 1. A method for identifying a ligand that selectively binds to a modified ligand-gated ion channel (LGIC), comprising: providing one or more candidate ligands to the modified LGIC of claim 1; detecting binding between the candidate ligand and the modified LGIC, thereby identifying a ligand that selectively binds to the modified LGIC; A method comprising:

115. The method of claim 114, wherein the modified LGIC is a chimeric LGIC comprising an LBD from a first LGIC and an IPD from a second LGIC.

116. 1. A method for detecting a modified ligand-gated ion channel (LGIC) comprising at least one modified LGIC subunit, comprising: providing one or more modified LGIC subunits according to claim 1; providing an agent that selectively binds to the modified LGIC; detecting binding between the modified LGIC and an agent that selectively binds to the modified LGIC, thereby detecting the modified LGIC; A method comprising:

117. The method of claim 116, wherein the agent that selectively binds to the modified LGIC comprises an antibody, a protein, or a small molecule.

118. 118. The method of claim 117, wherein the agent that selectively binds to the modified LGIC comprises a detectable label.

119. 119. The method of claim 118, wherein the detectable label comprises a label selected from the group consisting of a fluorescent label, a radioactive label, and a positron-emitting label.

120. A mammalian cell comprising the modified LGIC of claim 1.

121. A nucleic acid expressing the modified LGIC subunit of claim 1.

122. 1. A homomeric chimeric ligand-gated ion channel (LGIC) comprising a chimeric LGIC subunit, Each chimeric LGIC subunit is an alpha 7 nicotinic acetylcholine receptor ligand binding domain having a Q79G amino acid substitution and at least one of a W77F amino acid substitution, a Q139G amino acid substitution, a Y115F amino acid substitution, a G175K amino acid substitution, a Y210F amino acid substitution, a P216I amino acid substitution, a R27D amino acid substitution, and an E41R amino acid substitution; Glycine receptor ion pore domain and Including, wherein a ligand selected from the group consisting of tropisetron and granisetron selectively binds to the chimeric LGIC, and the chimeric LGIC minimally binds to acetylcholine (ACh). Homomeric chimeric ligand-gated ion channels (LGICs).

123. 1. A homomeric chimeric ligand-gated ion channel (LGIC) comprising a chimeric LGIC subunit, Each chimeric LGIC subunit is an alpha 7 nicotinic acetylcholine receptor ligand binding domain having an L131G amino acid substitution and at least one of a Q79S amino acid substitution, a Q139L amino acid substitution, a Y217F amino acid substitution, an R27D amino acid substitution, and an E41R amino acid substitution; an ion pore domain selected from the group consisting of a glycine receptor ion pore domain and a serotonin 3 receptor ion pore domain; Including, wherein a ligand selected from the group consisting of varenicline and tropisetron selectively binds to the chimeric LGIC, and the chimeric LGIC minimally binds to acetylcholine (ACh). Homomeric chimeric ligand-gated ion channels (LGICs).

124. 1. A method of treating a channelopathy in a mammal, comprising: administering the LGIC of claim 122 or 123 to a cell in a mammal; administering a ligand to said mammal; A method comprising:

125. 1. A method of modulating cellular excitability in a mammal, comprising: administering the LGIC of claim 122 or 123 to a cell in a mammal; administering a ligand to said mammal; A method comprising:

126. 1. A method of modulating cellular activity in a mammal, comprising: administering the LGIC of claim 122 or 123 to a cell in a mammal; administering a ligand to said mammal; A method comprising:

127. 1. A ligand with increased potency for modified ligand-gated ion channels (LGICs), comprising the formula VI: (In the formula, R1 is H or CH3; R2 is H, CH3, or an aromatic substituent; R3 is O or S. A ligand comprising:

128. 128. The ligand of claim 127, wherein said 7,8,9,10-tetrahydro-1H-6,10-methanoazepino[4,5-g]quinoxalin-2(6H)-one is selected from the group consisting of compound 0783, compound 0784, compound 0790, and compound 0792.

129. A ligand with increased potency for modified ligand-gated ion channels (LGICs), comprising 2-(pyridin-3-yl)-1,5,6,7,8,9-hexahydro-5,9-methanoimidazo[4',5':4,5]benzo[1,2-d]azepine (compound 0786): A ligand comprising:

130. The modified LGIC of claim 15, further comprising an endoplasmic reticulum transport sequence.

131. The modified LGIC of claim 130, wherein the endoplasmic reticulum transport sequence comprises the amino acid sequence: FCYENEV (SEQ ID NO: 16).

132. The modified LGIC of claim 131, comprising the amino acid sequence set forth in SEQ ID NO:

13.

133. The modified LGIC of claim 15, further comprising a CHRNB4 signal sequence.

134. The CHRNB4 signal sequence has the amino acid sequence:

134. The modified LGIC of claim 133, comprising:

135. The modified LGIC of claim 134, comprising the amino acid sequence set forth in SEQ ID NO:

14.

136. 16. The modified LGIC of claim 15, further comprising a KCNB1 somatic targeting sequence.

137. The KCNB1 somatic targeting sequence has the amino acid sequence:

137. The modified LGIC of claim 136, comprising:

138. The modified LGIC of claim 137, comprising the amino acid sequence set forth in SEQ ID NO:

15.

139. A synthetic nucleic acid construct comprising a nucleic acid sequence having at least 75% sequence identity with the sequence set forth in SEQ ID NO:

27.

140. The synthetic nucleic acid construct of claim 139, wherein the nucleic acid sequence having at least 75% sequence identity to the sequence set forth in SEQ ID NO: 27 comprises a nucleic acid sequence encoding a ligand binding domain (LBD) comprising an amino acid modification.

141. 141. The synthetic nucleic acid construct of claim 140, wherein said LBD is an alpha 7 nicotinic acetylcholine receptor (α7-nAChR) LBD and said modified LBD comprises an amino acid substitution at one or more amino acid residues selected from the group consisting of residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, and 219 of the α7-nAChR LBD.

142. The synthetic nucleic acid construct of claim 139, wherein the nucleic acid sequence having at least 75% sequence identity to the sequence set forth in SEQ ID NO: 27 comprises a nucleic acid sequence encoding an ion pore domain (IPD).

143. 143. The synthetic nucleic acid construct of claim 142, wherein the IPD is an IPD from a receptor selected from the group consisting of a serotonin 3 receptor (5HT3) IPD, a glycine receptor (GlyR) IPD, a gamma-aminobutyric acid (GABA) receptor IPD, and an alpha 7 nicotinic acetylcholine receptor (α7-nAChR) IPD.

144. 144. The synthetic nucleic acid construct of claim 143, wherein the IPD is a 5HT3 IPD.

145. A synthetic nucleic acid construct comprising a nucleic acid sequence having at least 75% sequence identity with the sequence set forth in SEQ ID NO:

28.

146. The synthetic nucleic acid construct of claim 145, wherein the nucleic acid sequence having at least 75% sequence identity to the sequence set forth in SEQ ID NO: 28 comprises a nucleic acid sequence encoding a ligand binding domain (LBD) comprising an amino acid modification.

147. 147. The synthetic nucleic acid construct of claim 146, wherein said LBD is an alpha 7 nicotinic acetylcholine receptor (α7-nAChR) LBD and said modified LBD comprises an amino acid substitution at one or more amino acid residues selected from the group consisting of residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, and 219 of the α7-nAChR LBD.

148. The synthetic nucleic acid construct of claim 145, wherein the nucleic acid sequence having at least 75% sequence identity to the sequence set forth in SEQ ID NO: 28 comprises a nucleic acid sequence encoding an ion pore domain (IPD).

149. 149. The synthetic nucleic acid construct of claim 148, wherein the IPD is an IPD from a receptor selected from the group consisting of a serotonin 3 receptor (5HT3) IPD, a glycine receptor (GlyR) IPD, a gamma-aminobutyric acid (GABA) receptor IPD, and an alpha 7 nicotinic acetylcholine receptor (α7-nAChR) IPD.

150. 150. The synthetic nucleic acid construct of claim 149, wherein the IPD is a GlyR IPD and the GlyR IPD comprises an amino acid modification.

151. 151. The synthetic nucleic acid construct of claim 150, wherein said modified GlyR IPD comprises an amino acid substitution at amino acid residue 298 of the GlyR IPD.

152. A synthetic nucleic acid construct comprising a nucleic acid sequence having at least 75% sequence identity with the sequence set forth in SEQ ID NO:

29.

153. The synthetic nucleic acid construct of claim 152, wherein the nucleic acid sequence having at least 75% sequence identity to the sequence set forth in SEQ ID NO: 29 comprises a nucleic acid sequence encoding a ligand binding domain (LBD) comprising an amino acid modification.

154. 154. The synthetic nucleic acid construct of claim 153, wherein said LBD is an alpha 7 nicotinic acetylcholine receptor (α7-nAChR) LBD and said modified LBD comprises an amino acid substitution at one or more amino acid residues selected from the group consisting of residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217, and 219 of the α7-nAChR LBD.

155. The synthetic nucleic acid construct of claim 152, wherein the nucleic acid sequence having at least 75% sequence identity to the sequence set forth in SEQ ID NO: 29 comprises a nucleic acid sequence encoding an ion pore domain (IPD).

156. 156. The synthetic nucleic acid construct of claim 155, wherein the IPD is an IPD from a receptor selected from the group consisting of a serotonin 3 receptor (5HT3) IPD, a glycine receptor (GlyR) IPD, a gamma-aminobutyric acid (GABA) receptor IPD, and an alpha 7 nicotinic acetylcholine receptor (α7-nAChR) IPD.

157. 157. The synthetic nucleic acid construct of claim 156, wherein the IPD is a GABA IPD and the GABA IPD comprises an amino acid modification.

158. 158. The synthetic nucleic acid construct of claim 157, wherein the modified GABA IPD comprises an amino acid substitution at amino acid residue 298 of the GABA IPD.

159. A synthetic nucleic acid construct comprising the sequence set forth in SEQ ID NO:

33.

160. A synthetic nucleic acid construct comprising the sequence set forth in SEQ ID NO:34.

Images