Modified ligand-gated ion channel and method of use

Abstract

To provide a material and a method for controlling ligand-gated ion channel (LGIC) activity.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). Also provided are an exogenous LGIC ligand that binds to modified LGIC and can activate it, a method for regulating ion transport traversing a membrane of a mammal cell, a method for regulating excitation in a mammal cell, and a method for treating a mammal having channelopathy.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Application No. 62 / 359,534, filed July 7, 2016, and the benefit of U.S. Application No. 62 / 486,779, filed April 18, 2017. 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 disclosure relates to materials and methods for controlling ligand-gated ion channel (LGIC) activity. For example, the present disclosure provides modified LGICs comprising at least one LGIC subunit with a modified ligand binding domain (LBD) and / or 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 a mammal with a channelopathy (e.g., a nerve channelopathy or a muscle channelopathy). In certain cases, the modified LGICs and exogenous LGIC ligands can be used to modulate (e.g., activate or inhibit) ion transport across a membrane of a mammalian cell. In certain cases, the modified LGICs and exogenous LGIC ligands can be used to modulate (e.g., increase or decrease) the excitability of a cell in a mammal. [Background technology]

[0003] 2. Background information Ion channels mediate ion flux within cells, which profoundly affects their biological functions. A prominent example of this is in neurons, where ion channels control the transmission of electrical signals between neurons to affect physiology, sensation, behavior, mood, and cognition.

[0004] Different LGICs have distinct ligand binding properties and specific 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. 1414 pp). For example, nicotinic acetylcholine receptors (nAChRs) bind the endogenous ligand acetylcholine (ACh), which activates a cation conductance, typically depolarizing the cell, 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 hyperpolarization and / or electrical shunting of 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 agonists such as ACh are not easily controlled. The present specification provides materials and methods for controlling 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 having an LBD and an IPD and including at least one modified LGIC subunit having at least one modified amino acid (e.g., an amino acid substitution). Also provided are exogenous LGIC ligands that can bind to and activate the modified LGIC. In certain cases, the modified LGICs and exogenous ligands can be used to treat a mammal having a channelopathy (e.g., a nerve channelopathy or a muscle channelopathy). In certain cases, the modified LGICs and exogenous LGIC ligands can be used to modulate (e.g., activate or inhibit) ion transport across the membrane of a mammalian cell. In certain cases, the modified LGICs and exogenous LGIC ligands can be used to modulate (e.g., increase or decrease) the excitability of a cell in a mammal.

[0007] Having the ability to control LGIC activity provides a unique and yet unrealized opportunity to achieve control of ion transport within cells. 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 delivery of exogenous LGIC ligands. For example, modified LGICs with reduced sensitivity to endogenous LGIC ligands prevent undesired activation of the modified LGIC and allow selective control over the modified LGIC by exogenous ligands. Furthermore, exogenous LGIC ligands with enhanced potency over the modified LGIC improve the selectivity of targeting of the modified LGIC to endogenous ion channels. Thus, the modified LCIGs and exogenous LGIC ligands provided herein are useful for achieving therapeutic effects while reducing side effects from small molecules against unintended targets.

[0008] As described herein, one or more mutations in modified LGIC can enhance the potency against exogenous LGIC ligands.Mutation of the α7 LBD of α7-GlyR at residue L131 (e.g., replacing Leu with Gly or Ala) reduced ACh potency against α7-GlyR (-6.4-fold) while increasing potency for varenicline (16-fold) and tropisetron (3.6-fold).Mutation of the α7 LBD of α7-GlyR at residue G175 (e.g., G175K) or P216 (e.g., P216I) enhanced potency against 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 from ACh (-8-fold). Furthermore, mutations in the α7 LBD at residues 77 (e.g., Trp replaced by Phe or Tyr) and / or 79 (e.g., Gln replaced by Gly, Ala, or Ser) and / or 131 (e.g., Leu replaced by Gly or Ala) and / or 141 (e.g., Leu replaced by 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 a mutation at residue 79 (e.g., Gln replaced with Gly), a mutation at residue 115 (e.g., Tyr replaced with Phe), and a mutation at residue 175 (e.g., Gly replaced with Lys) has more than 100-fold increased sensitivity to the exogenous tropane LGIC ligand Compound 723 (Tropane) and reduced ACh sensitivity (−15-fold) relative to the unmodified chimeric α7-GlyR LGIC.Furthermore, modified LGICs comprising at least one chimeric LGIC subunit with an α7 nAChR LBD (α7 LBD) with a mutation at residue 79 (e.g., replacing Gln with Ala, Gly, or Ser) and a GlyR IPD with a mutation at residue 298 (e.g., replacing Ala with Gly) have nearly 20-fold increased sensitivity to exogenous LGIC ligands such as quinuclidine or tropane. Further mutations at residues 27 (e.g., replacing Arg with Asp) and 41 (e.g., replacing Glu with Arg) of the α7 LBD reduced the association of the modified chimeric LGIC with unmodified ion channels. Further 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 LGICs to the endogenous ligand ACh. These chimeric LGICs allow for highly selective control of cellular functions in mammalian cells while minimizing cross-reactivity with endogenous signaling systems in mammals.

[0009] In general, one aspect of the present disclosure features a modified LGIC having at least one modified LGIC subunit comprising an LBD having an amino acid modification and an IPD, wherein an exogenous LGIC ligand activates the modified LGIC. The modified LGIC can be a chimeric LGIC having an LBD from a first LGIC and an IPD from a second LGIC. The LBD can be an alpha 7 nicotinic acetylcholine receptor (α7-nAChR) LBD. The modified LGIC of claim 3, wherein the 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 can be at residue 79 of the α7 LBD, and the amino acid substitution can be Q79A, Q79G, or Q79S. For example, the amino acid substitution at residue 79 of the α7 LBD can 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, and the GlyR IPD 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, and the GABA IPD 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 that includes an α7 LBD with a Q79G amino acid substitution, and a GlyR IPD with an A298G amino acid substitution. The exogenous LGIC ligand may 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 may 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 include 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 include at least one modified amino acid that confers increased ionic conductivity (conductance) to the modified LGIC. 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. If the at least one modified amino acid comprises an amino acid substitution at residue 217, the amino acid substitution can be a Y217F substitution. If the at least one modified amino acid comprises an amino acid substitution at residue 219, the amino acid substitution can be a D219A substitution.

[0011] In another aspect, the document features a ligand having increased potency for a modified ligand-gated ion channel (LGIC), where the ligand has Formula I: TIFF2025069252000001.tif31128 (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: TIFF2025069252000002.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: TIFF2025069252000003.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: TIFF2025069252000004.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. The 9-azabicyclo[3.3.1]nonane can be compound 0536.

[0015] In another aspect, the document features a ligand having increased potency for a modified ligand-gated ion channel (LGIC), where the ligand can be a 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine having formula V: TIFF2025069252000005.tif13128 (wherein R can be H or CH3, and A can be H or an aromatic substituent) The 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine can be varenicline, compound 0765, or compound 0770.

[0016] In another aspect, the document features a ligand having increased potency for modified ligand-gated ion channels (LGICs), where the ligand can be 1,4-diazabicyclo[3.2.2]nonane and has formula VI: TIFF2025069252000006.tif25128 (where R is H, F, or NO 2 (It can be The 1,4-diazabicyclo[3.2.2]nonane can be 3-(1,4-diazabicyclo[3.2.2]non-4-yl)dibenzo[b,d]thiophene 5,5-dioxide, compound 0773, or compound 0774.

[0017] In another aspect, the 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, where the exogenous LGIC ligand selectively binds to the modified LGIC. The modified LGIC has at least one modified LGIC subunit that includes an LBD and an IPD that includes at least one modified amino acid. The exogenous ligand is then administered to the mammal. The channelopathy may 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, paramyotonia congenita, retinitis pigmentosa, Timothy syndrome, tinnitus, seizures, trigeminal neuralgia, and multiple sclerosis.

[0018] In another aspect, the present specification features a method of modulating ion transport across a mammalian cell membrane. The method includes or consists essentially of administering a modified LGIC to a cell, where an exogenous LGIC ligand selectively binds to the modified LGIC. The modified LGIC has at least one modified LGIC subunit that includes an LBD and an IPD that includes 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, a glial cell, a muscle cell, a stem cell, an endocrine cell, or an 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.

[0019] In another aspect, the present specification features a method of modulating the excitability of a cell in a mammal. The method includes or consists essentially of administering a modified LGIC to a cell from the mammal, where an exogenous LGIC ligand selectively binds to the modified LGIC. The modified LGIC has at least one modified LGIC subunit that includes a LBD and an IPD that includes at least one modified amino acid. The exogenous ligand is then administered to the mammal. The modulation can include increasing the excitability of the cell or decreasing the excitability of the cell. The cell can be an excitable cell. The cell can be a neuron, a glial cell, a muscle cell, a stem cell, an endocrine cell, or an 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.

[0020] In another aspect, the present specification features a method of modulating the activity of a cell in a mammal. The method includes or consists essentially of administering a modified LGIC to a cell, where an exogenous LGIC ligand selectively binds to the modified LGIC. The modified LGIC has at least one modified LGIC subunit that includes an LBD and an IPD that includes at least one modified amino acid. The exogenous ligand is then administered to the mammal. The modulation can include increasing the activity of the cell or decreasing the activity of the cell. The activity can be ion transport, passive transport, excitation, inhibition, or exocytosis. The cell can be a neuron, a glial cell, a muscle cell, a stem cell, an endocrine cell, or an 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, a herpes simplex virus, or a lentivirus).

[0021] In another aspect, this document features a method for identifying a ligand that selectively binds to a modified LGIC. The method includes, 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.

[0022] In another aspect, the present specification features a method for detecting a modified LGIC. The method includes 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).

[0023] 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 discrepancy, the present specification, including definitions, will control.

[0024] [The present invention 1001] 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); A modified ligand-gated ion channel comprising: [The present invention 1002] The modified LGIC of the present invention 1001, which is a chimeric LGIC comprising a LBD from a first LGIC and an IPD from a second LGIC. [The present invention 1003] The modified LGIC of the present invention 1001, wherein said LBD is an alpha 7 nicotinic acetylcholine receptor (α7-nAChR) LBD. [The present invention 1004] 1003. The modified LGIC of the present invention, 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. [The present invention 1005] The modified LGIC of the present invention 1004, wherein said amino acid substitution is at residue 77 of the α7-nAChR LBD and said amino acid substitution is selected from the group consisting of W77F and W77Y. [The present invention 1006] The modified LGIC of the present invention 1004, wherein the amino acid substitution is at residue 79 of the α7-nAChR LBD and is selected from the group consisting of Q79A, Q79G, and Q79S. [The present invention 1007] 1004. The modified LGIC of the present invention, wherein the amino acid substitution is at residue 115 of the α7-nAChR LBD and is a Y115F substitution. [The present invention 1008] 1004. The modified LGIC of the present invention, 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. [The present invention 1009] The modified LGIC of the present invention 1004, wherein said amino acid substitution is at residue 139 of the α7-nAChR LBD and said amino acid substitution is selected from the group consisting of Q139G and Q139L. [The present invention 1010] 1004. The modified LGIC of the present invention, 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. [The present invention 1011] 1004. The modified LGIC of the present invention, wherein the amino acid substitution is at residue 210 of the α7-nAChR LBD and is a Y210F substitution. [The present invention 1012] The modified LGIC of the present invention 1004, wherein the amino acid substitution is at residue 216 of the α7-nAChR LBD and is a P216I substitution. [The present invention 1013] The modified LGIC of the present invention 1004, wherein the amino acid substitution is at residue 217 of the α7-nAChR LBD and the amino acid substitution is a Y217F substitution. [The present invention 1014] The modified LGIC of the present invention 1004, wherein the amino acid substitution is at residue 219 of the α7-nAChR LBD and is a D219A substitution. [The present invention 1015] The modified LGIC of the present invention 1004, wherein the α7-nAChR LBD comprises an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution. [The present invention 1016] The modified LGIC of the present invention 1004, wherein the α7-nAChR LBD comprises a L131M amino acid substitution and a Y115F amino acid substitution. [The present invention 1017] The modified LGIC of the present invention 1004, wherein the α7-nAChR LBD comprises a W77F amino acid substitution, a Q79G amino acid substitution, and a G175K amino acid substitution. [The present invention 1018] The modified LGIC of the present invention 1004, wherein the α7-nAChR LBD comprises a Q79G amino acid substitution, a Y115F amino acid substitution, and a G175K amino acid substitution. [The present invention 1019] The modified LGIC of the present invention 1004, wherein the α7-nAChR LBD comprises a Y115F amino acid substitution and a G175K amino acid substitution. [The present invention 1020] The modified LGIC of the present invention 1004, wherein the α7-nAChR LBD comprises a Q79G amino acid substitution and a 216I amino acid substitution. [The present invention 1021] The modified LGIC of the present invention 1001, wherein the IPD is an IPD from a receptor selected from the group consisting of serotonin 3 receptor (5HT3) IPD, glycine receptor (GlyR) IPD, gamma-aminobutyric acid (GABA) receptor IPD, and alpha 7 nicotinic acetylcholine receptor (α7-nAChR) IPD. [The present invention 1022] The modified LGIC of the present invention 1021, wherein said IPD comprises an amino acid substitution at residue 298. [The present invention 1023] The modified LGIC of the present invention 1022, wherein the IPD is a GlyR IPD and the amino acid substitution is an A298G substitution. [The present invention 1024] The modified LGIC of the present invention 1022, wherein the IPD is a GABA IPD and the amino acid substitution is a W298A substitution. [The present invention 1025] The modified LGIC of the present invention 1001, 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. [The present invention 1026] The modified LGIC of the present invention 1025, wherein the synthetic exogenous LGIC ligand is a tropane, and the tropane is selected from the group consisting of tropisetron, pseudo-tropisetron, nortropisetron, compound 723, compound 725, compound 737, and compound 745. [The present invention 1027] The modified LGIC of the present invention 1025, 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. [The present invention 1028] The modified LGIC of the present invention 1025, wherein the synthetic exogenous LGIC ligand is 9-azabicyclo[3.3.1]nonane, and the 9-azabicyclo[3.3.1]nonane is compound 536. [The present invention 1029] The modified LGIC of the present invention 1025, 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 765, and compound 770. [The present invention 1030] The modified LGIC of the present invention 1025, 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. [The present invention 1031] The modified LGIC of the present invention 1001, 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. [The present invention 1032] The modified LGIC of the present invention, wherein the unmodified LGIC is an endogenous LGIC. [The present invention 1033] The modified LGIC of the present invention 1032, wherein the endogenous LGIC is an endogenous α7-nAChR. [The present invention 1034] The modified LGIC of the present invention 1031, 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. [The present invention 1035] The modified LGIC of the present invention 1034, wherein said at least one modified amino acid comprises an R27D substitution and / or an E41R substitution. [The present invention 1036] The modified LGIC of the present invention, wherein said IPD is mouse 5HT3 IPD, and said mouse 5HT3 IPD further comprises at least one modified amino acid that confers increased ionic conductivity to said modified LGIC. [The present invention 1037] The modified LGIC of the present invention, wherein at least one modified amino acid in mouse 5HT3 IPD that confers increased ionic conductivity to said modified LGIC comprises an amino acid substitution at amino acid residue 425, 429, and / or 433 of mouse 5HT3 IPD. [The present invention 1038] The modified LGIC of the present invention 1037, wherein at least one modified amino acid comprises a R425Q substitution, a R429D substitution, and / or a R433A substitution. [The present invention 1039] The modified LGIC of the present invention, wherein said IPD is human 5HT3 IPD, and said human 5HT3 IPD further comprises at least one modified amino acid that confers increased ionic conductivity to said modified LGIC. [The present invention 1040] The modified LGIC of the present invention, wherein at least one modified amino acid in human 5HT3 IPD that confers increased ionic conductivity to said modified LGIC comprises an amino acid substitution at amino acid residue 420, 424, and / or 428 of human 5HT3 IPD. [The present invention 1041] The modified LGIC of the present invention 1040, wherein at least one modified amino acid comprises a R420Q substitution, a R424D substitution, and / or a R428A substitution. [The present invention 1042] 1001. The modified LGIC of the present invention, wherein said LBD has reduced binding to an endogenous LGIC ligand. [The present invention 1043] The modified LGIC of the present invention 1042, wherein the endogenous LGIC ligand is acetylcholine (ACh). [The present invention 1044] The modified LGIC of the present invention 1043, wherein the modified LGIC has an EC50 of more than 20 μM for Ach. [The present invention 1045] 1. A ligand having increased potency for modified ligand-gated ion channels (LGICs), comprising the compound of formula I: TIFF2025069252000007.tif32128(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: [The present invention 1046] The ligand of the present invention 1045, wherein said 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. [The present invention 1047] Formula II: TIFF2025069252000008.tif18128 (in the formula, X3 is O, NH, or CH2; Y is O or S; A is an aromatic substituent; R is H or pyridinylmethylene. The ligand of the present invention 1045 is a quinuclidine having the formula: [The present invention 1048] The ligand of the present invention 1047, wherein said 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. [The present invention 1049] The ligand of the present invention 1047, wherein said 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. [The present invention 1050] Formula III: TIFF2025069252000009.tif28128 (in the formula, X2 is NH or NMe; X3 is O, NH, or CH2; Y is O or S; A is an aromatic substituent. The ligand of the present invention 1045 is a tropane having the formula: [The present invention 1051] The ligand of the present invention 1050, 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. [The present invention 1052] The ligand of the present invention 1050, wherein said tropane is selected from the group consisting of tropisetron, pseudo-tropisetron, nortropisetron, compound 723, compound 725, compound 737, and compound 745. [The present invention 1053] Formula IV: TIFF2025069252000010.tif27128 (in the formula, X2 is NH or NMe; X3 is O, NH, or CH; Y is O or S; A is an aromatic substituent. The ligand of the present invention is 9-azabicyclo[3.3.1]nonane having the formula: [The present invention 1054] The ligand of the present invention 1054, wherein said aromatic substituent is selected from the group consisting of 4-chloro-benzene, 1H-indole, 1H-indazole, 7-methoxy-1H-indazole. [The present invention 1055] The ligand of the present invention 1054, 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. [The present invention 1056] 1. A ligand having increased potency for modified ligand-gated ion channels (LGICs), comprising the formula V: TIFF2025069252000011.tif13128 (in the formula, R is H or CH3; A is H or an aromatic substituent. A ligand comprising: [The present invention 1057] The ligand of the present invention 1056, wherein said 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. [The present invention 1058] 1. A ligand having increased potency for modified ligand-gated ion channels (LGICs), comprising the compound of formula VI: TIFF2025069252000012.tif25128 (wherein R is H, F, or NO 2 is) A ligand comprising: [The present invention 1059] The ligand of the present invention 1058, 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. [The present invention 1060] 1. A method of treating a channelopathy in a mammal, comprising: administering to a cell 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 Ion Pore Domain and administering the exogenous ligand to a mammal; A method comprising: [The present invention 1061] The 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 attacks), familial hemiplegic migraine, fibromyalgia, hyperkalemic periodic paralysis, hypokalemic periodic paralysis, Lambert-Eaton syndrome, on) myasthenic syndrome, long QT syndrome (e.g., Romano-Ward syndrome), short QT syndrome, malignant hyperthermia, mucolipidosis IV, myasthenia gravis, myotonia congenita, neuromyelitis optica, neuromyotonia, nonsyndromic hearing loss, paramyotonia congenita, retinitis pigmentosa, Timothy syndrome, tinnitus, seizures, trigeminal neuralgia, and multiple sclerosis. [The present invention 1062] 1. A method for modulating ion transport across a mammalian cell membrane, 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 Ion Pore Domain and administering the exogenous ligand to a mammal; A method comprising: [The present invention 1063] The method of claim 1062, wherein said modulation comprises activating ion transport. [The present invention 1064] The method of claim 1062, wherein said modulation comprises inhibiting ion transport. [The present invention 1065] The method of claim 1062, wherein said 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. [The present invention 1066] The method of claim 1062, wherein administering the modified LGIC to a cell comprises in vivo administration. [The present invention 1067] The method of claim 1062, wherein administering the modified LGIC to a cell comprises ex vivo administration. [The present invention 1068] 1. A method for modulating cellular excitability in a mammal, comprising: Administering to a cell from a 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 Ion Pore Domain and administering the exogenous ligand to a mammal; A method comprising: [The present invention 1069] The method of claim 1068, wherein said modulation comprises increasing the excitability of a cell. [The present invention 1070] The method of claim 1068, wherein said modulation comprises decreasing the excitability of a cell. [The present invention 1071] The method of claim 1068, wherein the cell is an excitable cell. [The present invention 1072] The method of claim 1068, wherein said 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. [The present invention 1073] The method of claim 1068, wherein administering the modified LGIC to a cell comprises in vivo administration. [The present invention 1074] The method of claim 1068, wherein administering the modified LGIC to a cell comprises ex vivo administration. [The present invention 1075] 1. A method for modulating a 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 Ion Pore Domain and administering the exogenous ligand to a mammal; A method comprising: [The present invention 1076] The method of claim 1075, wherein said modulation comprises increasing an activity of a cell. [The present invention 1077] The method of claim 1075, wherein said modulation comprises decreasing an activity of a cell. [The present invention 1078] The method of claim 1075, wherein said activity is selected from the group consisting of ion transport, passive transport, excitation, inhibition, and exocytosis. [The present invention 1079] The method of claim 1075, wherein said 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. [The present invention 1080] The method of claim 1075, wherein administering the modified LGIC to a mammalian cell comprises in vivo administration. [The present invention 1081] The method of claim 1075, wherein administering the modified LGIC to a mammalian cell comprises ex vivo administration. [The present invention 1082] The modified LGIC of any of 1060, 1062, 1068, or 1075, wherein the modified LGIC is a chimeric LGIC comprising a LBD from a first LGIC and an IPD from a second LGIC. [The present invention 1083] The method of claim 1082, wherein the chimeric LGIC is a homomeric chimeric LGIC. [The present invention 1084] The method of any of claims 1060, 1062, 1068, or 1075, wherein administering the modified LGIC to a cell comprises administering a nucleic acid encoding the modified LGIC. [The present invention 1085] The method of claim 1084, 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. [The present invention 1086] The method of claim 1085, 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. [The present invention 1087] The method of claim 1086, 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. [The present invention 1088] The method of the present invention, wherein the modified LGIC comprises a sequence as set forth in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:10. [The present invention 1089] The method of any one of claims 1060, 1062, 1068, or 1075, wherein said LBD is an alpha 7 nicotinic acetylcholine receptor (α7-nAChR) LBD. [The present invention 1090] 1089. The method of claim 1089, 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. [The present invention 1091] 1090. The method of claim 1090, wherein said amino acid substitution is at residue 77 of the α7-nAChR LBD, and said amino acid substitution is selected from the group consisting of W77F and W77Y. [The present invention 1092] 1090. The method of claim 1090, wherein said amino acid substitution is at residue 79 of the α7-nAChR LBD, and said amino acid substitution is selected from the group consisting of Q79A, Q79G, and Q79S. [The present invention 1093] 1090. The method of claim 1090, wherein the amino acid substitution is at residue 115 of the α7-nAChR LBD and the amino acid substitution is a Y115F substitution. [The present invention 1094] 1090. The method of claim 1090, wherein said amino acid substitution is at residue 131 of the α7-nAChR LBD, and said amino acid substitution is selected from the group consisting of L131A, L131G, L131M, and L131N. [The present invention 1095] 1090. The method of claim 1090, wherein the amino acid substitution is at residue 139 of the α7-nAChR LBD and is a Q139G or Q139L substitution. [The present invention 1096] 1090. The method of claim 1090, 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. [The present invention 1097] 1090. The method of claim 1090, wherein the amino acid substitution is at residue 210 of the α7-nAChR LBD and the amino acid substitution is a Y210F substitution. [The present invention 1098] The method of claim 1090, wherein the amino acid substitution is at residue 216 of the α7-nAChR LBD and the amino acid substitution is P216I. [This invention 1099] 1090. The method of claim 1090, wherein the amino acid substitution is at residue 217 of the α7-nAChR LBD and the amino acid substitution is Y217F. [The present invention 1100] The method of claim 1090, wherein said amino acid substitution is at residue 219 of the α7-nAChR LBD and the amino acid substitution is D219A. [The present invention 1101] The method of any of claims 1060, 1062, 1068, or 1075, wherein said IPD comprises at least one modified amino acid. [The present invention 1102] 1101. The method of claim 1101, wherein said 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. [The present invention 1103] 1102. The method of claim 1102, wherein said IPD is a GlyR IPD and the at least one modified amino acid comprises an amino acid substitution at residue 298 of modified LGIC. [The present invention 1104] 1103. The method of claim 1103, wherein the amino acid substitution at residue 298 of the modified LGIC is an A298G substitution. [The present invention 1105] The modified LGIC of the present invention 1102, wherein said IPD is a GABA IPD and at least one modified amino acid in the GABA IPD comprises an amino acid substitution at residue 298 of the modified LGIC. [The present invention 1106] The modified LGIC of the present invention 1105, wherein the amino acid substitution at residue 298 of said chimeric LGIC is a W298A substitution. [The present invention 1107] The modified LGIC of any of claims 1060, 1062, 1068, or 1075, wherein the exogenous LGIC ligand is a synthetic exogenous LGIC ligand. [The present invention 1108] 1107. The method of claim 1107, wherein said 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. [The present invention 1109] The method of claim 1108, 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. [The present invention 1110] The method of claim 1108, 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. [The present invention 1111] The method of claim 1108, wherein said synthetic exogenous LGIC ligand is 9-azabicyclo[3.3.1]nonane, and 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. [The present invention 1112] The method of claim 1108, wherein said synthetic exogenous LGIC ligand is 1,4-diazabicyclo[3.2.2]nonane, and said 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. [The present invention 1113] The method of claim 1108, 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 0765, and compound 0770. [The present invention 1114] 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 a modified LGIC of the present invention; detecting binding between the candidate ligand and the modified LGIC, thereby identifying a ligand that selectively binds to the modified LGIC; A method comprising: [The present invention 1115] The method of claim 1114, wherein said modified LGIC is a chimeric LGIC comprising a LBD from a first LGIC and an IPD from a second LGIC. [The present invention 1116] 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 of the present invention; 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: [The present invention 1117] The method of claim 1116, wherein the agent that selectively binds to the modified LGIC comprises an antibody, a protein, or a small molecule. [The present invention 1118] 1117. The method of claim 1117, wherein the agent that selectively binds to the modified LGIC comprises a detectable label. [The present invention 1119] The method of any one of claims 1118 to 1120, wherein said detectable label comprises a label selected from the group consisting of a fluorescent label, a radioactive label, and a positron emitting label. [The present invention 1120] A mammalian cell comprising a modified LGIC of the present invention. [The present invention 1121] A nucleic acid expressing a modified LGIC subunit of the invention. [The present invention 1122] A homomeric chimeric ligand-gated ion channel (LGIC) comprising chimeric LGIC subunits, each chimeric LGIC subunit comprising: 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; a glycine receptor ion pore domain; 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). [The present invention 1123] A homomeric chimeric ligand-gated ion channel (LGIC) comprising chimeric LGIC subunits, each chimeric LGIC subunit comprising: an alpha 7 nicotinic acetylcholine receptor ligand binding domain having a L131G amino acid substitution and at least one of a Q79S amino acid substitution, a Q139L amino acid substitution, a Y217F amino acid substitution, a 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). [The present invention 1124] 1. A method of treating a channelopathy in a mammal, comprising: administering a LGIC of the invention 1122 or 1123 to a cell in a mammal; administering a ligand to the mammal; A method comprising: [The present invention 1125] 1. A method for modulating cellular excitability in a mammal, comprising: administering a LGIC of the invention 1122 or 1123 to a cell in a mammal; administering a ligand to the mammal; A method comprising: [The present invention 1126] 1. A method for modulating a cellular activity in a mammal, comprising: administering a LGIC of the invention 1122 or 1123 to a cell in a mammal; administering a ligand to the mammal; A method comprising: 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 description of the drawings]

[0025] [Figure 1A] FIG. 1 shows an exemplary amino acid sequence of a chimeric LGIC. 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 some agonists. Mutations at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. When LBD mutations were combined with mutations at amino acid residue 298 of GlyR or GABAC IPD, potency was significantly enhanced. When α7 nAChR LBD mutations were combined with mutations at amino acid residues G175 and P216, potency was also enhanced. 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]FIG. 1 shows an exemplary amino acid sequence of a chimeric LGIC. 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 some agonists. Mutations at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. When LBD mutations were combined with mutations at amino acid residue 298 of GlyR or GABAC IPD, potency was significantly enhanced. When α7 nAChR LBD mutations were combined with mutations at amino acid residues G175 and P216, potency was also enhanced. 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] FIG. 1 shows an exemplary amino acid sequence of a chimeric LGIC. 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 some agonists. Mutations at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. When LBD mutations were combined with mutations at amino acid residue 298 of GlyR or GABAC IPD, potency was significantly enhanced. When α7 nAChR LBD mutations were combined with mutations at amino acid residues G175 and P216, potency was also enhanced. 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]FIG. 1 shows an exemplary amino acid sequence of a chimeric LGIC. 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 some agonists. Mutations at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. When LBD mutations were combined with mutations at amino acid residue 298 of GlyR or GABAC IPD, potency was significantly enhanced. When α7 nAChR LBD mutations were combined with mutations at amino acid residues G175 and P216, potency was also enhanced. D) Amino acid sequence of the α7-GABAc chimeric receptor (SEQ ID NO:10) comprising the human α7 nAChR LBD (SEQ ID NO:2) and human GABAC IPD (SEQ ID NO:9) constructs. [Figure 1E] FIG. 1 shows an exemplary amino acid sequence of a chimeric LGIC. Mutation of amino acid residue 77 (e.g., W77F or W77Y) resulted in sensitivity to granisetron and tropisetron. Mutation of amino acid residue 79 (e.g., Q79G) was the most effective for some agonists. Mutation of amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. When LBD mutations were combined with mutations of amino acid residue 298 of GlyR or GABAC IPD, potency was significantly enhanced. When α7 nAChR LBD mutations were combined with mutations at amino acid residues G175 and P216, potency was also enhanced. E) Amino acid sequence of rat nAChR sequence (SEQ ID NO: 12). [Diagram 2] Figure 2 shows the EC50 of tropisetron for the α7-5HT3 chimeric LGIC and mutants 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 (log scale). *P<0.05, indicating 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 4A] Figure 4 shows the relative potency of known nAChR agonists on the α7-GlyR chimeric LGIC. A) Graph of EC50 for Q79 LBD mutants normalized to unmodified α7-GlyR chimeric channels (log scale). [Figure 4B] Figure 4 shows the relative potency of known nAChR agonists on the α7-GlyR chimeric LGIC. B) Graph of the EC50 for the A298G IPD mutation normalized to the unmodified α7-GlyR chimeric channel (log scale). [Figure 4C] Figure 4 shows the relative potency of known nAChR agonists on α7-GlyR chimeric LGICs. C) Graph of EC50 for α7-GlyRA298G (log scale) normalized to the unmodified α7-GlyR chimeric channel and compared to the double mutant channel α7Q79G-GlyRA298G. *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 structure 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 association of the chimeric LCIG α7nAChR 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 charge-reversal mutations at the subunit interface. [Figure 6B] Figure 6 shows mutations that reduce the association of chimeric LCIG α7nAChR 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 α7nAChR LBD with the unmodified LBD. C) Association of α7-5HT3 LGIC with 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 co-expressing channels with HA and V5 epitopes, anti-HA IP followed by anti-V5 immunoblotting shows co-immunoprecipitation of each type of unmodified channel, but not the charge-reversal mutation in LBD α7R21D, E41R-5HT3-V5. The MW of α7-5HT3 is approximately 48 kD (arrows). [Figure 7]Figure 7 shows that chimeric LGICs can be controlled with 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 40 pA current injections (PRE) that are potently inhibited by 30 nM tropisetron. After washout of tropisetron (WASH), neuronal firing is restored. [Figure 8A] Figure 8 shows the activity of agonists against chimeric LGICs with the G175K mutation. A) Graph of EC50 for the Q79G G175K LBD mutant against known agonists (log scale), normalized to the unmodified α7-GlyR chimeric channel. [Figure 8B] Figure 8 shows the activity of agonists for chimeric LGICs with the G175K mutation. B) Graph of EC50 for ACh and tropisetron for channels with mutations in the α7-GlyR chimeric LGIC. The mutation resulting in a channel with high potency for tropisetron and low potency for the endogenous ligand acetylcholine (ACh) is optimal (grey shading). Unmod: unmodified α7-GlyR chimeric LGIC. [Figure 8C] Figure 8 shows the activity of agonists on chimeric LGICs carrying the G175K mutation. C) Action potentials of cortical neurons from mouse brains transduced with α7Q79G, Y115F, G175K-GlyR chimeric LGICs. Neurons fire in response to current injection (PRE) and are potently inhibited by 100 nM tropisetron. After washout of tropisetron (WASH), neuronal firing is restored. [Figure 9A] Figure 9 shows the activity of agonists on chimeric LGICs with the L131G mutation. A) Graph of EC50 for L131 LBD mutants against known agonists (log scale), normalized to the unmodified α7-GlyR chimeric channel. [Figure 9B]Figure 9 shows the activity of agonists on chimeric LGICs with the L131G mutation. B) Graph of EC50 for ACh and tropisetron for channels with mutations in the α7L131G-GlyR chimeric LGIC. [Figure 9C] Figure 9 shows agonist activity for chimeric LGICs 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 (grey shading). Unmod: unmodified α7-GlyR chimeric LGIC. [Figure 9D] Figure 9 shows the activity of agonists against chimeric LGICs carrying the L131G mutation. D) Action potentials of cortical neurons from mouse brains transduced with α7L131G, Q139L, Y217F-GlyR chimeric LGICs. Neurons fire in response to current injection (PRE) and are potently inhibited by 10 nM varenicline even at over 6-fold higher injected currents. After washout of tropisetron (WASH), neuronal firing is restored. [Figure 10] Figure 10 shows the chemical structures of LGIC agonists. A) Chemical structures of LGIC agonists with substitution patterns most compatible with potency enhancement for α7Q79G, Y115F, G175K-GlyR. B) Chemical structures of LGIC agonists with substitution patterns most compatible with potency enhancement for α7L131G, Q139L, Y217F-GlyR or α7L131G, Q139L, Y217F-5HT3 HC. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Detailed Description The present specification provides modified LGICs and methods of using the same. For example, the present specification provides modified LGICs that include at least one modified LGIC subunit having a LBD and an IPD and at least one modified amino acid (e.g., amino acid substitution). In certain cases, the modified LGIC can be a chimeric LGIC. For example, the chimeric LGIC can include a LBD from a first LGIC and an IPD from a second LGIC. In certain cases, the modified amino acid can confer pharmacological selectivity to the modified LGIC. For example, the modified amino acid can confer selective binding of an exogenous LGIC ligand to the modified LGIC. For example, the modified amino acid can confer reduced (minimized or eliminated) binding of an unmodified LGIC subunit (a LGIC subunit lacking the modification and / or an endogenous LGIC subunit) to the modified LGIC. For example, the modified amino acid can confer reduced (minimized or eliminated) binding of an endogenous LGIC ligand to the modified LGIC.

[0027] The modified LGICs provided herein can be used, for example, in methods for treating channel disorders (e.g., nerve or muscle channel disorders). For example, the modified LGICs and exogenous LGIC ligands that can bind to and activate the modified LGICs can be used to treat mammals with channel disorders. In certain cases, the modified LGICs and exogenous LGIC ligands can be used to regulate (e.g., activate or inhibit) ion transport across the membrane of a mammalian cell. In certain cases, the modified LGICs and exogenous LGIC ligands can be used to regulate (e.g., increase or decrease) the excitability of a cell in a mammal.

[0028] Qualified LGIC As used herein, a "modified" LGIC is a LGIC that includes at least one LGIC subunit. The modified LGIC subunit may include 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 subunit described herein may be a modification of a LGIC from any suitable species (e.g., human, rat, mouse, dog, cat, horse, cow, goat, pig, or monkey). In certain cases, the modified LGIC may 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.

[0029] The modified LGIC may be homomeric (e.g., having any number of the same modified LGIC subunits) or heteromeric (e.g., having at least one modified LGIC subunit and any number of different LGIC subunits). In certain cases, the modified LGIC described herein may be a homomeric modified LGIC. The modified LGIC described herein may include any suitable number of modified LGIC subunits. In certain cases, the modified LGIC may be a trimer, tetramer, pentamer, or hexamer. For example, the modified LGIC described herein may be a pentamer.

[0030] The modified LGIC subunits described herein can be any suitable modification of LGIC. LGICs can conduct anions, cations, or both through a cell membrane in response to binding of a ligand. For example, LGICs can conduct sodium (Na + ), 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 GABAA -ρ (also called GABAc) receptors, GlyR, 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 a 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 the nAChR can be an α7 LBD. A representative rat α7 nAChR amino acid sequence (including both the LBD and IPD) is as follows: TIFF2025069252000013.tif68158

[0031] In certain cases, the modified LGIC subunits described herein may include an LBD from an α7 nAChR. Examples of α7 nAChR LBDs include, but are not limited to, a human α7 nAChR LBD having the amino acid sequence set forth in SEQ ID NO:1, a human α7 nAChR LBD having the amino acid sequence set forth in SEQ ID NO:2, and a 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: TIFF2025069252000014.tif119158

[0032] In certain cases, the modified LGIC subunit described herein may comprise an IPD from a 5HT3 receptor. Examples of 5HT3 IPD 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. TIFF2025069252000015.tif76158

[0033] In certain cases, the modified LGIC subunit described herein may include 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. TIFF2025069252000016.tif33158

[0034] 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 called GABAc). A An example of a -ρ IPD is, but is not limited to, a human GABA receptor having the amino acid sequence set forth in SEQ ID NO:9. A -ρ IPD. In certain cases, GABA A -ρ IPD is the 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. TIFF2025069252000017.tif32158

[0035] When calculating the percentage of sequence identity, the two sequences are aligned and the number of identical matches of 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 value. It is understood that the length of the aligned region can be a portion of one or both sequences up to the full length size 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 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 and default parameters, which calculates the best match between the 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.

[0036] When the modified LGIC subunit described herein is a chimeric LGIC subunit, the chimeric LGIC subunit can comprise the LBD and IPD from the same species, or the LBD and IPD from different species.In certain cases, the chimeric LGIC subunit can comprise the LBD from human LGIC protein and the IPD from human LGIC protein.For example, the chimeric LGIC subunit can comprise the human α7 LBD and the human GlyR IPD.In certain cases, the chimeric LGIC subunit can comprise the LBD from human LGIC protein and the IPD from mouse LGIC protein.For example, the chimeric LGIC subunit can comprise the human α7 LBD and the mouse 5HT3 IPD.

[0037] When the modified LGIC subunit described herein is a chimeric LGIC subunit, the chimeric LGIC subunit may include various fusion points connecting the LBD and the IPD, such that the number of amino acids in the LBD may differ when the LBD is fused to different IPDs to form a chimeric channel subunit. 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 Figure 1A and Figure 1C with Figure 1B).

[0038] The modified LGIC subunits described herein may include a LBD having at least one modified amino acid and / or an IPD having at least one modified amino acid. For example, the modified LGIC subunits described herein may include 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 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 may include a GlyR IPD having 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 having an amino acid substitution at amino acid residue 298 of the α7-GABAc chimeric receptor (e.g., SEQ ID NO:10). In certain cases, the modified LGIC subunit described herein may include more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more) amino acid modifications. 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 (a 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.

[0039] In certain embodiments, the modified LGIC subunits described herein may include at least one modified amino acid that confers selective binding (e.g., enhanced binding or enhanced efficacy) to the modified LGIC with an exogenous LGIC ligand. The binding to the exogenous LGIC ligand may be more selective than the binding to the endogenous LGIC ligand. The modified LGIC subunits having selective binding to the exogenous LGIC ligand may include any suitable LDB (e.g., α7 LBD). In certain embodiments, the modified LGIC subunits may include 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 as shown 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 an alanine (e.g., Q79A), a glycine (e.g., Q79G), or a 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), 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). The modified LGIC subunit having selective binding to an exogenous LGIC ligand can be any suitable IPD (e.g., GlyR IPD or GABA. A In certain aspects, the modified LGIC subunit may comprise a GlyR IPD set forth in SEQ ID NO:5, and 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 may be substituted with an amino acid residue such as glycine (e.g., A298G). In certain aspects, the modified LGIC subunit may comprise a GABA IPD set forth in SEQ ID NO:9, and 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 may be substituted with an amino acid residue such as glycine (e.g., A298G). A -ρIPD, the amino acid modification of which may be α7-GABA A The -ρ chimeric receptor (e.g., SEQ ID NO: 10) may be substituted at amino acid residue 298. In certain cases, the tryptophan at amino acid residue 298 of SEQ ID NO: 10 may be substituted with an amino acid residue such as alanine (e.g., W298A).

[0040] 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).

[0041] 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 may also be described as having enhanced potency for the exogenous ligand. In certain cases, modified LGIC subunits described herein that selectively bind to an exogenous LGIC ligand may have enhanced potency for the exogenous ligand of at least 4-fold (e.g., at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold). In certain cases, modified LGIC subunits described herein that selectively bind to exogenous LGIC ligands may have enhanced potency for the exogenous ligand of 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 may have enhanced potency for the exogenous ligand of about 10-fold to about 100-fold. For example, a modified LGIC subunit described herein that selectively binds to an exogenous LGIC ligand can have about 10-fold to about 20-fold enhanced potency for the exogenous ligand.

[0042] In certain embodiments, the modified LGIC subunits described herein may include at least one modified amino acid that confers to the modified LGIC reduced (e.g., minimized or eliminated) binding to 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 a LGIC subunit lacking a modification that confers to the modified LGIC reduced binding to unmodified LGIC subunits, or the unmodified LGIC may be an endogenous LGIC subunit. The modification that confers to the modified LGIC reduced binding to unmodified LGIC subunits may be a charge reversal modification. The modified LGIC subunit with reduced binding to unmodified LGIC subunits may include any suitable LBD (e.g., α7 LBD). In certain embodiments, the modified LGIC subunit may include an α7 LBD as 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 an 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 an arginine (e.g., E41R). In certain cases, the modified LGIC subunits described herein can include an α7 LBD having an R27D substitution and an E41R.

[0043] In certain embodiments, the modified LGIC subunits described herein may include at least one modified amino acid that confers to the modified LGIC a reduced (e.g., minimized or eliminated) binding of an endogenous LGIC ligand. The endogenous LGIC ligand may be ACh. The modified LGIC subunit with reduced binding of an endogenous LGIC ligand may include any suitable IPD (e.g., GlyR LBD). For example, the modified LGIC subunit may include an α7 LBD as shown 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 a 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 replaced 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 replaced 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 replaced 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 a 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 an alanine (e.g., D219A).

[0044] In certain embodiments, the modified LGIC subunits described herein may include at least one modified amino acid that confers an increased ion conductance to the modified LGIC. In certain cases, the modified LGIC subunits may include a 5HT3 IPD as set forth in SEQ ID NO:3, and the amino acid modification may be a substitution at amino acid residues 425, 429, and / or 433. The modified LGIC subunits described herein may include a 5HT3 IPD having an R425Q substitution, an R429D substitution, and an R433A substitution. In certain cases, the modified LGIC subunits may include a 5HT3 IPD as set forth in SEQ ID NO:4, and the amino acid modification may be a substitution at amino acid residues 420, 424, and / or 428. The modified LGIC subunits described herein may include a 5HT3 IPD having an R420Q substitution, an R424D substitution, and an R428A substitution.

[0045] In certain cases, the modified LGIC described herein may include 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).

[0046] In certain cases, the modified LGIC described herein may include 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 Q139G amino acid substitutions, and a mouse 5HT3 IPD (SEQ ID NO:3).

[0047] 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) having a Q79G amino acid substitution and a Y115F amino acid substitution, and a human GlyR IPD (SEQ ID NO: 5) having an A298G amino acid substitution.

[0048] 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) having a Q79G amino acid substitution and a Q139G amino acid substitution, and a human GlyR IPD (SEQ ID NO: 5) having an A298G amino acid substitution.

[0049] 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) having an R27D, E41R, Q79G, and Y115F amino acid substitution, and a human GlyR IPD (SEQ ID NO: 5) having an A298G amino acid substitution.

[0050] In certain cases, the modified LGIC described herein may include 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).

[0051] 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).

[0052] 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).

[0053] 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).

[0054] 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).

[0055] 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) having a substitution at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N), and a human 5HT3 IPD (SEQ ID NO:4).

[0056] In certain cases, the modified LGIC described herein may include 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).

[0057] 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) having 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) having R420Q, R424D, and R428A substitutions.

[0058] 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) having 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) having R420Q, R424D, and R428A substitutions.

[0059] In certain cases, the modified LGIC described herein may include 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).

[0060] In certain cases, the modified LGIC described herein may include 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).

[0061] In certain cases, the modified LGIC described herein may include 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).

[0062] In certain cases, the modified LGIC described herein may include 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).

[0063] 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).

[0064] In certain cases, the modified LGIC described herein may include 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).

[0065] Where 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 substitutions of R425Q, R429D, and R433A in mouse 5HT3 IPD correspond to substitutions of R420Q, R424D, and R428A in human 5HT3 IPD.

[0066] Any method can be used to obtain the modified LGIC subunits described herein. In certain cases, peptide synthesis methods can be used to generate the modified LGIC subunits described herein. Examples of peptide synthesis methods include, but are not limited to, liquid phase peptide synthesis and solid phase peptide synthesis. In certain cases, protein biosynthesis methods can be used to generate 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 with essentially the same modifications and / or essentially the same amino acid sequence) will self-assemble through interactions between LBDs to form modified LGIC.

[0067] The present specification also provides nucleic acids encoding modified LGIC subunits described herein, as well as constructs for expressing nucleic acids encoding modified LGIC subunits described herein (e.g., plasmids, non-viral vectors, viral vectors (e.g., adeno-associated virus, herpes simplex virus, or lentiviral vectors, etc.)). The nucleic acids encoding modified LGIC subunits described herein can be operably linked to any suitable promoter. The promoter can be a natural (i.e., minimal) promoter or a composite promoter. The promoter can be a ubiquitous (i.e., constitutive) promoter or a regulatable promoter (e.g., inducible, tissue-specific, cell type-specific (e.g., neuronal 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, CAMKII, CMV, CAG, enolase, TRPV1, POMC, NPY, AGRP, MCH, and orexin promoters. In certain cases, the nucleic acids encoding modified LGIC subunits described herein can be operably linked to a neuron-specific promoter.

[0068] The present specification also provides a cell (e.g., a mammalian cell) having a modified LGIC described herein. A mammalian cell having a modified LGIC described herein can be obtained by any suitable method. In certain cases, a preassembled 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.

[0069] LGIC Ligand The present specification also provides a LGIC ligand that can bind to and activate the modified LGIC described herein. The LGIC ligand that can bind to and activate the modified LGIC described herein can be exogenous or endogenous. The LGIC ligand that can bind to and activate the modified LGIC described herein can be naturally occurring or synthetic. The LGIC ligand that can bind to and activate the modified LGIC described herein can be canonical or non-canonical. The LGIC ligand that can bind to and activate the modified LGIC described herein can be an agonist or an antagonist. 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, 10A, and 10B).

[0070] A LGIC ligand that can bind and activate a modified LGIC described herein may have selective binding (e.g., enhanced binding or increased potency) for the modified LGIC described herein. In certain cases, a LGIC ligand that can bind and activate a modified LGIC described herein does not bind to and activate an endogenous receptor. A LGIC ligand that selectively binds 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, a LGIC ligand that selectively binds to and activates the modified LGIC may have enhanced potency relative to the modified LGIC by about 10-fold to about 300-fold (e.g., about 10-fold to about 250-fold, about 10-fold to about 200-fold, about 10-fold to about 150-fold, about 10-fold to about 100-fold, about 25-fold to about 300-fold, about 50-fold to about 300-fold, about 100-fold to about 300-fold, about 200-fold to about 300-fold, about 25-fold to about 250-fold, about 50-fold to about 200-fold, or about 100-fold to about 150-fold). In certain cases, a 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, a 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, a 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, a LGIC ligand (e.g., tropisetron) binds to a modified LGIC subunit described herein (e.g., α7). Q79G -GlyR A298G For example, a 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, the 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, greater than 65 μM, greater than 80 μM, or greater than 100 μM) for a modified LGIC subunit described herein. For example, the 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, greater than 65 μM, greater than 80 μM, or greater than 100 μM) for a modified LGIC subunit described herein. Q79G、Y115F -GlyR A298G ) may have an EC50 of greater than 100 μM.

[0071] In certain embodiments, the LGIC ligand can be a synthetic ligand capable of binding and activating the modified LGIC described herein, and can be a quinuclidine, a tropane, a 9-azabicyclo[3.3.1]nonane, or a 2-phenyl-7,8,9,10-tetrahydro-6H-6,10-methanoazepino[4,5-g]quinoxaline.

[0072] LGIC ligands capable of binding to and activating the modified LGICs described herein have formula I: TIFF2025069252000018.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; 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.

[0073] A LGIC ligand capable of binding to and activating the modified LGIC described herein can be quinuclidine. Quinuclidine has the formula II: TIFF2025069252000019.tif18128 (wherein X3 can be O, NH, or CH2; Y can be O or S; A can be an aromatic substituent; 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).

[0074] A LGIC ligand capable of binding to and activating the modified LGIC described herein can be a tropane. A tropane has formula III: TIFF2025069252000020.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).

[0075] The LGIC ligand capable of binding to and activating the modified LGIC described herein can be 9-azabicyclo[3.3.1]nonane. 9-azabicyclo[3.3.1]nonane has formula IV: TIFF2025069252000021.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, is 4-chloro-benzene. Examples of 9-azabicyclo[3.3.1]nonane 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).

[0076] In certain cases, the LGIC ligand can be a 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, represented by formula V: TIFF2025069252000022.tif13128 (wherein R is H or CH 3 and A is H or an aromatic substituent. 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, and compound 0770 (see, e.g., FIG. 10A, Table 3, and Table 9).

[0077] In certain cases, the LGIC ligand can be 1,4-diazabicyclo[3.2.2]nonane, represented by formula VI: TIFF2025069252000023.tif25128 (where R=H, F, NO 2 ) Examples of 1,4-diazabicyclo[3.2.2]nonanes include, but are not limited to, 3-(1,4-diazabicyclo[3.2.2]nonane-4-yl)dibenzo[b,d]thiophene 5,5-dioxide, compound 0773, and compound 0774 (see, e.g., FIG. 10B, Tables 6, and 9).

[0078] How to use The present specification also provides methods of using the modified LGICs described herein and LGIC ligands that can bind to and activate the modified LGICs described herein. The LGIC ligands that can bind to and activate the modified LGICs can be used to activate the modified LGICs with temporal and / or spatial control based on delivery of the ligand.

[0079] 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 LGICs described herein and detecting binding between the candidate ligands and the modified LGIC.

[0080] 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.

[0081] In certain aspects, the modified LGICs described herein and LGIC ligands capable of binding and activating the modified LGICs described herein can be used to treat a mammal having a channelopathy (e.g., a nerve channelopathy or a muscle channelopathy). For example, a mammal having a channelopathy can be treated by administering a modified LGIC described herein and then administering a LGIC ligand capable of binding and activating the modified LGIC. For example, a mammal having a channelopathy 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 amino acid substitution, an E41R amino acid substitution, a Q79G amino acid substitution, and a Y115F amino acid substitution, and a human GlyR IPD (SEQ ID NO:5) with an A298G amino acid substitution), and then administering 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 a 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.

[0082] Any type of mammal can be treated with the modified LGIC described herein and the LGIC ligand that can bind and activate the modified LGIC described herein.For example, humans and other primates such as monkeys can be treated with the modified LGIC described herein and the LGIC ligand that can bind and activate the modified LGIC described herein.In certain cases, dogs, cats, horses, cows, pigs, sheep, rabbits, mice, and rats can be treated with the modified LGIC described herein and the LGIC ligand that can bind and activate the modified LGIC described herein.

[0083] Any suitable method can be used to identify a mammal having and / or at risk of developing a channelopathy. For example, genetic testing can be used to identify a mammal having and / or at risk of developing a channelopathy.

[0084] Once a mammal has been identified as having a channelopathy and / or at risk for developing a channelopathy, the mammal may be administered or instructed to self-administer a modified LGIC as described herein, and then administered or instructed to self-administer a LGIC ligand capable of binding to and activating the modified LGIC as described herein. The modified LGIC as described herein and the LGIC ligand capable of binding to and activating the modified LGIC as described herein may be administered together or may be administered separately.

[0085] When the materials and methods described herein are used to treat mammals having and / or 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 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, paramyotonia congenita, 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.

[0086] In certain cases, the modified LGICs described herein and LGIC ligands that can bind and activate the modified LGICs described herein can be used to modulate a cellular activity. The cellular activity modulated using the modified LGICs described herein and LGIC ligands that can bind and activate the modified LGICs described herein can be any cellular activity. Examples of cellular activities 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. The cellular activity can be increased or decreased. For example, the modified LGICs described herein and LGIC ligands that can bind and activate the modified LGICs described herein can be used to modulate (e.g., increase) ion transport across the membrane of a cell. For example, the modified LGICs described herein and LGIC ligands that can bind and activate the modified LGICs described herein can be used to modulate (e.g., increase) the excitability of a cell.

[0087] The modified LGICs described herein and the LGIC ligands that can bind 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.

[0088] 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 preassembled 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 with 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.

[0089] The modified LGIC described herein can be detected (e.g., to confirm its presence in a cell) by any suitable method. 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 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 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 a cell can include fluorescent imaging, autoradiography, functional MRI, PET, and SPECT.

[0090] The modified LGICs described herein and LGIC ligands capable of binding 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 the modified LGICs described herein and LGIC ligands capable of binding and activating the modified LGICs described herein and treating with acetazolamide, dichlorophenamide, mexilitine, glucose, calcium gluconate, L-DOPA, muscle stimulation, spinal cord stimulation, brain stimulation, and / or nerve stimulation.

[0091] In embodiments where the modified LGICs described herein and LGIC ligands capable of binding 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 at the same time or independently. For example, the modified LGICs described herein and LGIC ligands capable of binding and activating the modified LGICs described herein can be administered first and the one or more additional agents can be administered second, or vice versa. In embodiments where the modified LGICs described herein and LGIC ligands capable of binding 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 and activating the modified LGICs described herein. For example, the modified LGICs described herein and LGIC ligands capable of binding and activating the modified LGICs described herein can be administered before, during, or after the administration of the one or more additional therapies.

[0092] In certain cases, the modified LGICs described herein and / or LGIC ligands capable of binding and activating the modified LGICs described herein can be formulated into pharma- ceutically 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 and activating the modified LGICs described herein can be formulated together with one or more pharma- ceutical acceptable carriers (additives) and / or diluents. The pharmaceutical compositions can be formulated for administration in solid or liquid form, including, but not limited to, sterile solutions, suspensions, sustained release formulations, tablets, capsules, pills, powders, and granules.

[0093] 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.

[0094] Pharmaceutical compositions comprising 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 administration), or inhalation administration. When administered orally, pharmaceutical compositions comprising 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 that 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 that may contain suspending agents and thickening agents. Compositions for inhalation can be delivered, for example, using an inhaler, nebulizer, and / or dry powder inhaler. The formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and can 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 can be prepared from sterile powders, granules, and tablets.

[0095] A pharma- ceutically 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 a LGIC ligand capable of binding 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 a LGIC ligand capable of binding and activating a modified LGIC described herein can be administered systemically by intravenous or oral administration to a mammal (e.g., a human), or by inhalation therethrough. 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 a LGIC ligand capable of binding and activating a modified LGIC described herein can be administered locally by transdermal, subcutaneous, intramuscular, intracranial, or open surgical administration (e.g., injection) to a target tissue of a mammal (e.g., a human).

[0096] 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.

[0097] The frequency of administration may be any frequency that ameliorates symptoms of the channelopathy without causing significant toxicity to the mammal. For example, the frequency of administration may be from about once a week to about three times a day, from about twice a month to about six times a day, or from about twice a week to about once a day. The frequency of administration may remain constant or may be variable during the treatment period. 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 a LGIC ligand capable of binding to and activating a modified LGIC described herein may include a rest period. For example, 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 a LGIC ligand capable of binding to and activating a modified LGIC described herein may be administered daily for two weeks, followed by a two-week rest period, and such a regimen may be repeated multiple times. As with the effective amount, various factors may affect the actual frequency of administration 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 frequency of administration.

[0098] The effective period of 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 may vary from days to weeks, months, or years. In certain cases, the effective period for treating a channelopathy may range from about one month to about ten years. Multiple factors affect the actual effective period used for a particular treatment. For example, the effective period may 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.

[0099] In certain instances, the progress of treatment and the condition of the mammal being treated for the channelopathy can be monitored. Any suitable method can be used to monitor the symptoms of the channelopathy.

[0100] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0101] Example 1: Potency-enhancing ligand-binding domain mutations A screen was performed using a panel of 41 α7-5HT3 chimeric channels with mutated LBDs 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 revealed 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 gave 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 were potent α7 Q79G Furthermore, nortropisetron and PNU-282987 each showed significantly improved efficacy against α7 Q79A -5HT3 and α7 Q79SIn general, agonists based on quinuclidine or tropane pharmacophores with linked aromatic structures that interact with the complementary binding face 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.

[0102] Table 1: Potency of nAChR agonists against chimeric cation channels mutated at Gln79 in HEK cells. Mean EC50, SEM in parentheses (μM). TIFF2025069252000024.tif54157

[0103] 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 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 α7 Q79G -GlyR was the most preferred mutant chimeric l-channel.

[0104] Another association observed in the small molecule screen was that mutations at Trp77 increased the expression of α7 W77F -5HT3 (EC50: 1.2 μM), α7 W77Y -5HT3 (EC50: 1.1 μM), and α7 W77FThe results showed that the drug granisetron exerted agonistic activity at the α7-5HT3 or α7-GlyR (EC50: 0.66 μM) receptors. Granisetron is a 5HT3 receptor antagonist, which does not activate the α7-5HT3 or α7-GlyR.

[0105] 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.

[0106] 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 virtually ligand-free open probability, which makes them unsuitable for ligand-controlled manipulation of cells. 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. For the standard agonists ACh, nicotine, and epibatidine, as well as for varenicline and tropisetron, α7-GlyR A298G Agonist potency was not significantly enhanced in α7 nAChRs. 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 was not as effective as a mutation in the LBD, depending on the ligand structure.

[0107] The Q79G mutation in the LBD and the A298G IPD mutation in the α7-GlyR were examined (Table 2). Q79G -GlyR A298G resulted in a synergistic enhancement of potency for α7 nAChR agonists, showing up to an 18-fold potency enhancement for the α7-GlyR. The enhancement from this double mutant channel was greater than the enhancement from the individual mutations for agonists RS56812, tropisetron, nortropisetron, and PNU-282987. Further highlighting the unexpected structural sensitivity of this mutation combination, multiple agonists such as ACh, nicotine, epibatidine, varenicline, and cytisine enhanced potency at both the α7-GlyR and α7 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 for some, but not all, nicotinic agonists by approximately 10-20 fold.

[0108] Table 2: Potency of nAChR agonists against mutant chimeric chloride channels. Mean EC50 and SEM in parentheses for agonist activity in HEK cells expressing chimeric channels (μM). TIFF2025069252000025.tif59170

[0109] 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.

[0110] 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. In addition, we also tested the known α7 nAChR agonist PHA-543613 (Walker et al. 2006, Wishka et al. 2006) and demonstrated enhanced potency at α7. Q79G -GlyR A298G These molecules generally showed 10- to 100-fold enhanced potency (Table 3), which suggests that for these pharmacophores, a specific range of structural features are involved in the α7 Q79G -GlyR A298G This shows that the effect is consistent with improved efficacy.

[0111] These results indicate that the modified LGICs can be activated by synthetic quinuclidine- and tropane-containing LGIC ligands.

[0112] 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. TIFF2025069252000026.tif233125

[0113] Example 4: Mutations that reduce acetylcholine responsiveness The α7 nAChR is relatively insensitive to ACh compared to 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 substantially reduced acetylcholine responsiveness to >100 μM in certain cases, and α7 Q79G、Y115F -5HT3, α7 Q79G、Q139G -5HT3, α7 Q79G、Q139G -GlyR A298, α7 Q79G、Y115F -GlyR A298G For α7, it only modestly reduced the potency of certain agonists. Q79G、Y115F -GlyR A298G has an EC50 of 13 nM for nortropisetron and >100 μM for ACh (Table 4).

[0114] Table 4: Potency of nAChR agonists against mutant chimeric chloride channels with reduced acetylcholine responsiveness. Mean EC50 and SEM in μM for activity in HEK cells expressing the chimeric channels. TIFF2025069252000027.tif48161

[0115] 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.

[0116] 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 LBD-to-LBD interactions (Celie et al 2004 Neuron 41: 907-14). To minimize unwanted association with endogenous α7 nAChR subunits and / or unwanted association of chimeric channels, potential intersubunit bridges were identified by inspecting the crystal structure of an acetylcholine-binding protein and identifying nearby intersubunit residues with opposite charges that also have 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 retain 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 (Figure 6B). Co-expression of these charge-reversed channels with the α7-5HT3 channel, which has the unmodified sequence, showed that the charge-reversed subunits did not coimmunoprecipitate with the unmodified channel (Figure 6C). R27D、E41R、Q79G、Y115F -GlyR A298G Combination of potency-enhancing and acetylcholine-blocking mutations to obtain agonists revealed that some agonists retained high potency relative to their cognate agonists (Table 4, right column).

[0117] 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.

[0118] Example 6: LBD mutations that increase ligand potency Gly of the α7 nAChR LBD in the α7-GlyR chimeric channel 175 and Pro 216The mutations in Gly 175 to Lys (α7 G175K -GlyR) showed increased potency against ACh (5-fold) (Table 5). G175K It was also found that for the -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-GlyR, a greater than 10-fold increase in potency was observed over α7-GlyR for the clinically used drugs tropisetron, varenicline, cytisine, granisetron, and epibatidine. P216I For -GlyR, the potency enhancement was approximately 3-fold (Table 5).

[0119] (Table 5) Enhanced agonist potency by G175K and P216I mutations in a7GlyR chimeric channels. TIFF2025069252000028.tif232113nd=Not decided

[0120] 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 seen 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 considerably reduced, but enhanced potency for granisetron was observed (Table 5).

[0121] To develop a channel with reduced ACh responsiveness but high potency to other agonists, we targeted the α7 G175K -GlyR was combined with further mutations that increased the potency of certain agonists. Combination with W77F reduced ACh potency and α7 W77F、G175K α7-GlyR showed increased potency over α7-GlyR for granisetron, nortopisetron, and tropisetron, but not for PNU282-987, varenicline, cytisine, or PHA-543613 (Table 5). Combination of G175K with Q79G reduced ACh potency and increased potency of 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).

[0122] Further reductions in ACh potency were achieved by incorporating mutations at W77F, Q79G, L141F, Y115F, G175K, and Y210F in various combinations, 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、G175KThere was also enhanced responsiveness to other tropane and quinuclidine core structures, particularly quinuclidine thioureas 702 and 703, and tropane esters 723, 725, 726, 736, 737, 738, and 745, 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 -GlyR reduced ACh responsiveness while maintaining high potency responses to tropisetron and nortropisetron (Table 5). W77F、Q79G、G175K The α7-GlyR also showed enhanced potency for additional tropane-based core structures, such as compounds 723 and 725, as well as the clinically used drugs mequitazine and promazine (Table 6). W77F、G175K、Y210F -GlyR reduced ACh responsiveness but significantly increased potency to granisetron (Table 5). L141F、Y115F、G175K The α7-GlyR reduced ACh responsiveness while conferring sensitivity to granisetron (Table 5). Q79G、Q139L、G175K -GlyR reduced ACh responsiveness but exhibited a robust response to nortropisetron (Table 5).

[0123] Table 6. Potency enhancement of tropanes, quinuclidine agonists, 9-azabicyclo[3.3.1]nonane agonists, diazabicyclo[3.2.2]nonane agonists, and promazine by G175K and P216I α7GlyR chimeric channels. Indole and indazole aromatic (A) substituents attached at the 3-position. TIFF2025069252000029.tif235138TIFF2025069252000030.tif235148nd=Not determined; brackets:SEM

[0124] α7 G175K -GlyR and α7 P216I-GlyR was matched with the non-associating mutations R27D, E41R, and the GlyR IPD mutation A298G, which further enhanced ligand potency for granisetron, epibatidine, varenicline, cytisine, PNU-282987, tropisetron, nortropisetron, and PHA-543613, along with mutations at Q79G, Y115F, and G175K (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).

[0125] Table 7. G175K and A298G mutations in α7GlyR chimeric channels and α7GABAc (GABA A Enhancement of agonist efficacy by W298A at the -ρ (also called ρ) channel. TIFF2025069252000031.tif79157nd=Not determined;Bracket:SEM

[0126] α7 Y115F Gly of the α7 nAChR LBD in the α7-GlyR chimeric channel 175 Further amino acid substitutions in α7 also enhanced agonist potency. Y115F The potency of tropisetron in -GlyR chimeric channels 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).

[0127] (Table 8) Enhanced agonist potency by G175 mutation in α7GlyR Y115F chimeric channel. TIFF2025069252000032.tif49148nd=Not determined;Bracket:SEM

[0128] 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 -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-GlyR showed a similar potency enhancement for varenicline relative to α7-GlyR (21-fold), but also reduced 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) (FIG. 9B and Table 9). L131G、Q139L、Y217F -GlyR also showed extremely high potency for compound 770 (0.001 μM), compound 773 (0.00034 μM), and compound 774 (0.00013 μM) (FIG. 10). 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 for 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 The -5HT3 HC (Table 9) improved the efficacy of varenicline by 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).

[0129] Table 9. Enhanced agonist potency by chimeric channels with the L131 mutation. TIFF2025069252000033.tif235122nd=Not determined;Bracket:SEM

[0130] Example 7: Chimeric LGICs in neurons α7 Q79G -GlyR A298G Or the A7 Q79G、Y115F、G175K AAV or DNA plasmids containing nucleic acids encoding the -GlyR chimeric LGIC were transduced into mouse cortical neurons. Low concentrations of tropisetron (30 nM or 100 nM) were administered to mouse cortical neurons. Neuronal activity was suppressed by application of low concentrations of the agonist (Figures 7 and 8C).

[0131] α7 L131G、Q139L、Y217F A DNA plasmid containing a nucleic acid encoding the -GlyR chimeric LGIC was transfected into mouse cortical neurons. A low concentration of varenicline (10 nM) was administered to the mouse cortical neurons. Neuronal activity was suppressed by application of a low concentration of the agonist (Figure 9C).

[0132] These results indicate that modulating LGIC activity can be controlled in neurons using low concentrations of the LGIC ligands, tropisetron and varenicline.

[0133] Example 8: Chimeric LGICs in therapy Chemical genetic tools provide an attractive strategy for combining drug and gene therapy because the use of exogenously delivered ion channels selectively engaged in the administration of drugs allows cellular function to be modulated in a consistent manner across different cell types in a variety of indications using the same ion channel and ligand. 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.

[0134] For the drug tropisetron, we found that it binds the α7 receptor with an EC50 of 11 nM, similar to the IC50 of 10 nM tropisetron reported for its therapeutic target, the 5HT3 receptor. Q79G -GlyR A298G (Combrink et al. 2009 Pharmacological reports: PR 61: 785-97).

[0135] 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.

[0136] Sequence information SEQUENCE LISTING <110> Howard Hughes Medical Institute <120> MODIFIED LIGAND-GATED ION CHANNELS AND METHODS OF USE <150> US 62 / 359,534 <151> 2016-07-07 <150> US 62 / 486,779 <151> 2017-04-18 <160> 12 <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> 236 <212> PRT <213> Humana <400> 4 Leu Phe Tyr Val Val Ser Leu Leu Leu Pro Ser Ile Phe Leu Met Val 1 5 10 15 Met Asp Ile Val Gly Phe Tyr Leu Pro Pro Asn Ser Gly Glu Arg Val 20 25 30 Ser Phe Lys Ile Thr Leu Leu Leu Gly Tyr Ser Val Phe Leu Ile Ile 35 40 45 Val Ser Asp Thr Leu Pro Ala Thr Ala Ile Gly Thr Pro Leu Ile Gly 50 55 60 Val Tyr Phe Val Val Cys Met Ala Leu Leu Val Ile Ser Leu Ala Glu 65 70 75 80 Thr Ile Phe Ile Val Arg Leu Val His Lys Gln Asp Leu Gln Gln Pro 85 90 95 Val Pro Ala Trp Leu Arg His Leu Val Leu Glu Arg Ile Ala Trp Leu 100 105 110 Leu Cys Leu Arg Glu Gln Ser Thr Ser Gln Arg Pro Pro Ala Thr Ser 115 120 125 Gln Ala Thr Lys Thr Asp Asp Cys Ser Ala Met Gly Asn His Cys Ser 130 135 140 His Met Gly Gly Pro Gln Asp Phe Glu Lys Ser Pro Arg Asp Arg Cys 145 150 155 160 Ser Pro Pro Pro Pro Pro Pro Arg Glu Ala Ser Leu Ala Val Cys Gly Leu 165 170 175 Leu Gln Glu Leu Ser Ser Ile Arg Gln Phe Leu Glu Lys Arg Asp Glu 180 185 190 Ile Arg Glu Val Ala Arg Asp Trp Leu Arg Val Gly Ser Val Leu Asp 195 200 205 Lys Leu Leu Phe His Ile Tyr Leu Leu Ala Val Leu Ala Tyr Ser Ile 210 215 220 Thr Leu Val Met Leu Trp Ser Ile Trp Gln Tyr Ala 225 230 235 <210> 5 <211> 202 <212> PRT <213> mouse <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 Serves Only Lys Gly Only Asn Asn Serves 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> (alpha)7-5HT3 chimeric receptor <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> (alpha)7-GlyR chimeric receptor <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> (alpha)7-5HT3 chimeric receptor <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> (alpha)7- GABAC chimeric receptor <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

Claims

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 ion pore domain (IPD); 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. 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. 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. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 216 of the α7-nAChR LBD and is a P216I substitution.

13. 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. 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 an 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 serotonin 3 receptor (5HT3) IPD, glycine receptor (GlyR) IPD, gamma-aminobutyric acid (GABA) receptor IPD, and alpha 7 nicotinic acetylcholine receptor (α7-nAChR) IPD.

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. 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, pseudo-tropisetron, 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 quinuclidine, the quinuclidine being 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 residue 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 a R425Q substitution, a R429D substitution, and / or a 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 residue 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. The modified LGIC of claim 42, wherein the endogenous LGIC ligand is acetylcholine (ACh).

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 having 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 said tropane is selected from the group consisting of tropisetron, pseudo-tropisetron, 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 having increased potency for modified ligand-gated ion channels (LGICs), comprising the formula V: (In the formula, R is H or CH3; A is H or an aromatic substituent. A ligand comprising:

57. 57. The ligand of claim 56, wherein said 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.

58. 1. A ligand with increased potency for modified ligand-gated ion channels (LGICs), comprising the formula VI: (Wherein, R is H, F, or NO. ２ (It 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 a cell 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 Ion 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, paramyotonia congenita, retinitis pigmentosa, Timothy syndrome, tinnitus, seizures, trigeminal neuralgia, and multiple sclerosis.

62. 1. A method for modulating ion transport across a mammalian cell membrane, comprising: Administering to a cell 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 Ion 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 the 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. The method of claim 62, wherein administering the modified LGIC to a cell comprises in vivo administration.

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

68. 1. A method for modulating cellular excitability in a mammal, comprising: Administering to a cell from a 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 Ion 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 cellular excitability.

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

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

75. 1. A method for modulating a 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 Ion 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 a cell.

77. 76. The method of claim 75, wherein the modulation comprises decreasing an activity of a 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. The method of claim 75, wherein administration of the modified LGIC to a mammalian cell comprises in vivo administration.

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

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

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

10.

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

105. The modified LGIC of claim 102, wherein the IPD is a GABA IPD and 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. 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. 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. The method of claim 108, 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 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. 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. A homomeric chimeric ligand-gated ion channel (LGIC) comprising chimeric LGIC subunits, each chimeric LGIC subunit comprising: 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 a E41R amino acid substitution; a glycine receptor ion pore domain; 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. A homomeric chimeric ligand-gated ion channel (LGIC) comprising chimeric LGIC subunits, each chimeric LGIC subunit comprising: an alpha 7 nicotinic acetylcholine receptor ligand binding domain having a L131G amino acid substitution and at least one of a Q79S amino acid substitution, a Q139L amino acid substitution, a Y217F amino acid substitution, a R27D amino acid substitution, and a 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 the mammal; A method comprising:

125. 1. A method for 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 the mammal; A method comprising:

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

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