Modified Ligand-Gated Ion Channels and Methods of Use

Abstract

The present specification relates to materials and methods for regulating ligand-gated ion channel (LGIC) activity. For example, modified LGICs are provided, including at least one LGIC subunit with a modified ligand binding domain (LBD) and / or a modified ion pore domain (IPD). Also provided are exogenous LGIC ligands capable of binding to and activating the modified LGICs, as well as methods for regulating ion transport across the membrane of a mammalian cell, regulating cellular excitability in a mammal, and treating a mammal with a channel disorder.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 62 / 359,534, filed July 7, 2016, and U.S. Patent 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 specification relates to materials and methods for controlling ligand-gated ion channel (LGIC) activity. For example, the present specification provides modified LGICs including at least one LGIC subunit having a modified ligand binding domain (LBD) and / or a modified ion pore domain (IPD). Also provided are exogenous LGIC ligands capable of binding to and activating the modified LGIC. In certain cases, the modified LGICs and exogenous ligands can be used to treat mammals with channelopathies (e.g., nerve channelopathies or muscle channelopathies). In certain cases, the modified LGICs and exogenous LGIC ligands can be used to regulate (e.g., activate or inhibit) ion transport across membranes of mammalian cells. In certain cases, the modified LGICs and exogenous LGIC ligands can be used to regulate (e.g., increase or decrease) cellular excitability in mammals. [Background technology]

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

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

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

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

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

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

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

[0010] In another aspect, the present disclosure features a modified LGIC having at least one modified LGIC subunit comprising an LBD and an IPD with at least one modified amino acid, wherein the at least one modified amino acid in the LBD reduces binding to an endogenous LGIC ligand. The modified LGIC can be a chimeric LGIC having an LBD from a first LGIC and an IPD from a second LGIC. The endogenous LGIC ligand can be ACh. The modified LGIC can have an EC50 of greater than 20 μM for ACh. The at least one modified amino acid can include an amino acid substitution at residues 115, 139, 210, 217, and / or 219. When the at least one modified amino acid includes an amino acid substitution at residue 115, the amino acid substitution can be a Y115F substitution. When the at least one modified amino acid includes an amino acid substitution at residue 139, the amino acid substitution can be a Q139G or Q139L substitution. When the at least one modified amino acid includes an amino acid substitution at residue 210, the amino acid substitution can be a Y210F substitution. If at least one modified amino acid comprises an amino acid substitution at residue 217, the amino acid substitution can be a Y217F substitution. If 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, this document features a ligand with increased potency for a modified ligand-gated ion channel (LGIC), wherein the ligand has a structure represented by Formula I: TIFF0007755922000001.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: TIFF0007755922000002.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: TIFF0007755922000003.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: TIFF0007755922000004.tif27128 (wherein X1 can be CH, X2 can be NH or NMe, X3 can be O, NH, or CH, Y can be O or S, and A can be an aromatic substituent) The aromatic substituent can be 4-chloro-benzene. 9-Azabicyclo[3.3.1]nonane can be compound 0536.

[0015] In another aspect, this document features a ligand with increased potency for modified ligand-gated ion channels (LGICs), where the ligand can be a 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine having formula V: TIFF0007755922000005.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, this document features a ligand with 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: TIFF0007755922000006.tif25128 (where R can be H, F, or NO2) The 1,4-diazabicyclo[3.2.2]nonane can be 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-dioxide, compound 0773, or compound 0774.

[0017] In another aspect, this document features a method for treating a channelopathy in a mammal. The method includes, or consists essentially of, administering a modified LGIC to a cell in the mammal, wherein an exogenous LGIC ligand selectively binds to the modified LGIC. The modified LGIC has at least one modified LGIC subunit including an LBD and an IPD that include at least one modified amino acid. The exogenous ligand is then administered to the mammal. The channelopathy can be Bartter syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT), congenital hyperinsulinemia, cystic fibrosis, Dravet syndrome, episodic ataxia, erythromelalgia, generalized epilepsy (e.g., with febrile seizures), familial hemiplegic migraine, fibromyalgia, hyperkalemic periodic paralysis, hypokalemic periodic paralysis, Lambert-Eaton myasthenic syndrome, long QT syndrome (e.g., Romano-Ward syndrome), short QT syndrome, malignant hyperthermia, mucolipidosis type IV, myasthenia gravis, myotonia congenita, neuromyelitis optica, neuromyotonia, nonsyndromic hearing loss, congenital paramyotonia, retinitis pigmentosa, Timothy syndrome, tinnitus, seizures, trigeminal neuralgia, and multiple sclerosis.

[0018] In another aspect, the present disclosure features a method for modulating ion transport across a mammalian cell membrane. The method comprises or consists essentially of administering a modified LGIC to a cell, wherein an exogenous LGIC ligand selectively binds to the modified LGIC. The modified LGIC has at least one modified LGIC subunit comprising an LBD and an IPD that include at least one modified amino acid. The exogenous ligand is then administered to the mammal. The modulation can include activating or inhibiting ion transport. The cell can be a neuron, glial cell, muscle cell, stem cell, endocrine cell, or immune cell. The administration of the modified LGIC to the cell can be in vivo or ex vivo. The administration of the modified LGIC to the cell can include administration of a nucleic acid encoding the modified LGIC.

[0019] In another aspect, the present disclosure features a method for modulating the excitability of a cell in a mammal. The method comprises or consists essentially of administering a modified LGIC to a cell derived from the mammal, wherein an exogenous LGIC ligand selectively binds to the modified LGIC. The modified LGIC has at least one modified LGIC subunit comprising an LBD and an IPD that include at least one modified amino acid. The exogenous ligand is then administered to the mammal. The modulation can include increasing or decreasing the excitability of the cell. The cell can be an excitable cell. The cell can be a neuron, glial cell, muscle cell, stem cell, endocrine cell, or immune cell. The administration of the modified LGIC to the cell can be in vivo or ex vivo. The administration of the modified LGIC to the cell can include administration of a nucleic acid encoding the modified LGIC.

[0020] In another aspect, the description features a method for modulating cellular activity in a mammal. The method comprises or consists essentially of administering a modified LGIC to a cell, wherein an exogenous LGIC ligand selectively binds to the modified LGIC. The modified LGIC has at least one modified LGIC subunit comprising an LBD and an IPD that include at least one modified amino acid. The exogenous ligand is then administered to the mammal. The modulation can include increasing or decreasing cellular activity. The activity can be ion transport, passive transport, excitation, inhibition, or exocytosis. The cell can be a neuron, glial cell, muscle cell, stem cell, endocrine cell, or immune cell. The administration of the modified LGIC to the cell can be in vivo or ex vivo. The administration of the modified LGIC to the cell can include administration of a nucleic acid encoding the modified LGIC (e.g., via a viral vector such as an adeno-associated virus, herpes simplex virus, or lentivirus).

[0021] In another aspect, this document features a method for identifying a ligand that selectively binds to a modified LGIC. The method comprises, or consists essentially of, providing one or more candidate ligands to a modified LGIC described herein and detecting binding between the candidate ligands and the modified LGIC, thereby identifying a ligand that selectively binds to the modified LGIC. The modified LGIC can be a homomeric modified LGIC.

[0022] In another aspect, the description features a method for detecting a modified LGIC. The method comprises, or consists essentially of, providing one or more modified LGIC subunits described herein, providing an agent that selectively binds to the modified LGIC, and detecting the modified LGIC by detecting binding between the modified LGIC and the agent that selectively binds to the modified LGIC. The agent that selectively binds to the modified LGIC can be an antibody, a protein (e.g., bungarotoxin), or a small molecule (e.g., a positron emission tomography (PET) ligand). The agent that selectively binds to the modified LGIC can include a detectable label (e.g., a fluorescent label, a radioactive label, or a positron-emitting label).

[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 conflict, 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); 1. A modified ligand-gated ion channel comprising: [The present invention 1002] 1001. A modified LGIC of the present invention, which is a chimeric LGIC comprising an LBD from a first LGIC and an IPD from a second LGIC. [The present invention 1003] 1001. The modified LGIC of the present invention, 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 said 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] 1004. The modified LGIC of the present invention, wherein said amino acid substitution is at residue 77 of the α7-nAChR LBD, and wherein said amino acid substitution is selected from the group consisting of W77F and W77Y. [The present invention 1006] 1004. The modified LGIC of the present invention, 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 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] 1004. The modified LGIC of the present invention, 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] 1004. The modified LGIC of the present invention, wherein the amino acid substitution is at residue 216 of the α7-nAChR LBD and is a P216I substitution. [The present invention 1013] 1004. The modified LGIC of the present invention, wherein the amino acid substitution is at residue 217 of the α7-nAChR LBD and is a Y217F substitution. [The present invention 1014] 1004. The modified LGIC of the present invention, wherein the amino acid substitution is at residue 219 of the α7-nAChR LBD, and the amino acid substitution is a D219A substitution. [The present invention 1015] 1004. The modified LGIC of the present invention, 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] 1004. The modified LGIC of the present invention, wherein said α7-nAChR LBD comprises a L131M amino acid substitution and a Y115F amino acid substitution. [The present invention 1017] 1004. The modified LGIC of the present invention, 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] 1004. The modified LGIC of the present invention, 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] 1004. The modified LGIC of the present invention, wherein said α7-nAChR LBD comprises a Y115F amino acid substitution and a G175K amino acid substitution. [The present invention 1020] 1004. The modified LGIC of the present invention, wherein said 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 a serotonin 3 receptor (5HT3) IPD, a glycine receptor (GlyR) IPD, a gamma-aminobutyric acid (GABA) receptor IPD, and an alpha 7 nicotinic acetylcholine receptor (α7-nAChR) IPD. [The present invention 1022] 1021. The modified LGIC of the present invention, 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, 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] 1025. The modified LGIC of the present invention, 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] 1001. The modified LGIC of the present invention, 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 1031, wherein said 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] 1031. The modified LGIC of the present invention, 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. [This invention 1035] 1034. The modified LGIC of the present invention, wherein said at least one modified amino acid comprises an R27D substitution and / or an E41R substitution. [The present invention 1036] 1001. The modified LGIC of the present invention, wherein the IPD is 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. [This invention 1037] 1036. 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 an R425Q substitution, an R429D substitution, and / or an R433A substitution. [This invention 1039] 1001. 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] 1039. 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. [This invention 1041] The modified LGIC of the present invention 1040, wherein at least one modified amino acid comprises an R420Q substitution, an R424D substitution, and / or an 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. [This invention 1043] The modified LGIC of the present invention 1042, wherein the endogenous LGIC ligand is acetylcholine (ACh). [This invention 1044] The modified LGIC of the present invention 1043, wherein the modified LGIC has an EC50 of more than 20 μM for Ach. [This invention 1045] 1. A ligand with increased potency for modified ligand-gated ion channels (LGICs), comprising the compound of formula I: TIFF0007755922000007.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] 1045. The ligand of the present invention, 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. [This invention 1047] Formula II: TIFF0007755922000008.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: [This invention 1048] 1047. The ligand of the present invention, 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. [This 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: TIFF0007755922000009.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 is a tropane having the formula: [This invention 1051] 1050. A ligand according to claim 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. [This 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. [This invention 1053] Formula IV: TIFF0007755922000010.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 1045 is 9-azabicyclo[3.3.1]nonane having the formula: [This 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. [This 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 with increased potency for modified ligand-gated ion channels (LGICs), comprising the compound of formula V: TIFF0007755922000011.tif13128 (In the formula, R is H or CH3; A is H or an aromatic substituent. A ligand comprising: [This invention 1057] The ligand of claim 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. [This invention 1058] 1. A ligand with increased potency for modified ligand-gated ion channels (LGICs), comprising the compound of formula VI: TIFF0007755922000012.tif25128 (wherein R is H, F, or NO) 2 is) A ligand comprising: [This 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 cells in the mammal a modified ligand-gated ion channel (LGIC), wherein an exogenous LGIC ligand selectively binds to the modified LGIC, the modified LGIC comprising at least one modified LGIC subunit, the modified LGIC subunit comprising: a ligand-binding domain comprising at least one modified amino acid; and Ion Pore Domain and administering the exogenous ligand to a mammal; A method comprising: [This invention 1061] The channelopathy may be 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 syndrome, on) 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. [This 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 Ionic Pore Domain and administering the exogenous ligand to a mammal; A method comprising: [The present invention 1063] 1063. The method of claim 1062, wherein said modulation comprises activating ion transport. [This invention 1064] 1063. The method of claim 1062, wherein said modulation comprises inhibiting ion transport. [This invention 1065] 1063. 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] 1062. The method of claim 1062, wherein administering the modified LGIC to a cell comprises in vivo administration. [This invention 1067] 1063. The method of claim 1062, wherein administering said modified LGIC to a cell comprises ex vivo administration. [The present invention 1068] 1. A method of 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: [This invention 1069] 1068. The method of claim 1068, wherein said modulation comprises increasing the excitability of the cell. [The present invention 1070] 1068. The method of claim 1068, wherein said modulation comprises decreasing the excitability of the cell. [This invention 1071] The method of claim 1068, wherein said cell is an excitable cell. [This invention 1072] 1068. 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. [This invention 1073] 1068. The method of claim 1068, wherein administering the modified LGIC to a cell comprises in vivo administration. [This invention 1074] 1068. The method of claim 1068, wherein administering said modified LGIC to a cell comprises ex vivo administration. [This invention 1075] 1. A method of modulating cellular activity in a mammal, comprising: administering to the cell a modified ligand-gated ion channel (LGIC), wherein the exogenous LGIC ligand selectively binds to the modified LGIC, the modified LGIC comprising at least one modified LGIC subunit, the modified LGIC subunit comprising: a ligand-binding domain comprising at least one modified amino acid; and Ion Pore Domain and administering the exogenous ligand to a mammal; A method comprising: [This invention 1076] 1075. The method of claim 1075, wherein said modulation comprises increasing an activity of a cell. [This invention 1077] 1075. The method of claim 1075, wherein said modulation comprises decreasing an activity of a cell. [This invention 1078] 1075. The method of claim 1075, wherein said activity is selected from the group consisting of ion transport, passive transport, excitation, inhibition, and exocytosis. [This invention 1079] 1075. 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. [This invention 1081] 1075. The method of claim 1075, wherein administering the modified LGIC to a mammalian cell comprises ex vivo administration. [This invention 1082] The modified LGIC of any of 1060, 1062, 1068, or 1075, wherein said modified LGIC is a chimeric LGIC comprising an LBD from a first LGIC and an IPD from a second LGIC. [This invention 1083] The method of claim 1082, wherein the chimeric LGIC is a homomeric chimeric LGIC. [This invention 1084] 1076. The method of any of claims 1060, 1062, 1068, or 1075, wherein administering said modified LGIC to a cell comprises administering a nucleic acid encoding said modified LGIC. [This invention 1085] 1084. The method of claim 1084, wherein said 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. [This invention 1086] 1085. The method of claim 1085, wherein said 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. [This invention 1087] 1086. 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, SEQ ID NO:10. [This invention 1088] 1087. The method of claim 1087, wherein the modified LGIC comprises a sequence set forth in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10. [This invention 1089] 1076. The method of any 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. [This 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. [This 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. [This 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. [This 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. [This invention 1095] 1090. The method of claim 1090, 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. [This 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. [This 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. [This invention 1098] 1090. 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] 1090. The method of claim 1090, wherein the amino acid substitution is at residue 219 of the α7-nAChR LBD and the amino acid substitution is D219A. [The present invention 1101] 1076. 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] 1103. The method of claim 1102, wherein said IPD is a GlyR IPD and wherein the at least one modified amino acid comprises an amino acid substitution at residue 298 of modified LGIC. [The present invention 1104] 1104. The method of claim 1103, wherein the amino acid substitution at residue 298 of said modified LGIC is an A298G substitution. [This invention 1105] 1102. The modified LGIC of the present invention, wherein said IPD is a GABA IPD, and wherein 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] 1105. The modified LGIC of the present invention, wherein the amino acid substitution at residue 298 of said chimeric LGIC is a W298A substitution. [This invention 1107] The modified LGIC of any of 1060, 1062, 1068, or 1075, wherein the exogenous LGIC ligand is a synthetic exogenous LGIC ligand. [This 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. [This 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 said synthetic exogenous LGIC ligand is quinuclidine, and 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 702. [The present invention 1111] 1108. 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] 1108. 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] 1108. The method of claim 1108, wherein said synthetic exogenous LGIC ligand is 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, and 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. [This 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: [This invention 1115] 1114. The method of claim 1114, wherein said modified LGIC is a chimeric LGIC comprising an 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, the method 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: [This invention 1117] 1116. The method of claim 1116, wherein the agent that selectively binds to the modified LGIC comprises an antibody, a protein, or a small molecule. [This invention 1118] 1117. The method of claim 1117, wherein the agent that selectively binds to the modified LGIC comprises a detectable label. [This invention 1119] 1118. The method of claim 1118, 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. [This invention 1121] A nucleic acid expressing a modified LGIC subunit of the present invention. [This invention 1122] A homomeric chimeric ligand-gated ion channel (LGIC) comprising chimeric LGIC subunits, wherein each chimeric LGIC subunit is: an alpha 7 nicotinic acetylcholine receptor ligand binding domain having a Q79G amino acid substitution and at least one of a W77F amino acid substitution, a Q139G amino acid substitution, a Y115F amino acid substitution, a G175K amino acid substitution, a Y210F amino acid substitution, a P216I amino acid substitution, a R27D amino acid substitution, and an E41R amino acid substitution; 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). [This invention 1123] A homomeric chimeric ligand-gated ion channel (LGIC) comprising chimeric LGIC subunits, wherein each chimeric LGIC subunit is: 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, an R27D amino acid substitution, and an E41R amino acid substitution; an ion pore domain selected from the group consisting of a glycine receptor ion pore domain and a serotonin 3 receptor ion pore domain; Including, wherein a ligand selected from the group consisting of varenicline and tropisetron selectively binds to the chimeric LGIC, and the chimeric LGIC minimally binds to acetylcholine (ACh). Homomeric chimeric ligand-gated ion channels (LGICs). [This invention 1124] 1. A method of treating a channelopathy in a mammal, comprising: administering an LGIC of the invention 1122 or 1123 to cells in a mammal; administering a ligand to the mammal; A method comprising: [Invention 1125] 1. A method of modulating cellular excitability in a mammal, comprising: administering an LGIC of the invention 1122 or 1123 to cells in a mammal; administering a ligand to the mammal; A method comprising: [The present invention 1126] 1. A method of modulating cellular activity in a mammal, comprising: administering an LGIC of the invention 1122 or 1123 to cells 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 explanation of the drawings]

[0025] [Figure 1A]Figure 1 shows exemplary amino acid sequences of chimeric LGICs. Mutations at amino acid residue 77 (e.g., W77F or W77Y) conferred sensitivity to granisetron and tropisetron. Mutations at amino acid residue 79 (e.g., Q79G) were most effective against several agonists. Mutations at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. Potency was significantly enhanced when LBD mutations were combined with mutations at amino acid residue 298 of the GlyR or GABAC IPD. Potency was also enhanced when α7 nAChR LBD mutations were combined with mutations at amino acid residues G175 and P216. A) Amino acid sequence of the α7-5HT3 chimeric receptor (SEQ ID NO:6) comprising the human α7 nAChR LBD (SEQ ID NO:1) and mouse 5HT3 IPD (SEQ ID NO:3) constructs. [Figure 1B] Figure 1 shows exemplary amino acid sequences of chimeric LGICs. Mutations at amino acid residue 77 (e.g., W77F or W77Y) conferred sensitivity to granisetron and tropisetron. Mutations at amino acid residue 79 (e.g., Q79G) were most effective against several agonists. Mutations at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. Potency was significantly enhanced when LBD mutations were combined with mutations at amino acid residue 298 of the GlyR or GABAC IPD. Potency was also enhanced when α7 nAChR LBD mutations were combined with mutations at amino acid residues G175 and P216. B) Amino acid sequence of the α7-GlyR chimeric receptor (SEQ ID NO: 7), comprising the human α7 nAChR LBD (SEQ ID NO: 2) and human GlyR IPD (SEQ ID NO: 5) constructs. [Figure 1C]Figure 1 shows exemplary amino acid sequences of chimeric LGICs. Mutations at amino acid residue 77 (e.g., W77F or W77Y) conferred sensitivity to granisetron and tropisetron. Mutations at amino acid residue 79 (e.g., Q79G) were most effective against several agonists. Mutations at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. Potency was significantly enhanced when LBD mutations were combined with mutations at amino acid residue 298 of the GlyR or GABAC IPD. Potency was also enhanced when α7 nAChR LBD mutations were combined with mutations at amino acid residues G175 and P216. C) Amino acid sequence of the α7-5HT3 chimeric receptor (SEQ ID NO:8) comprising the human α7 nAChR LBD (SEQ ID NO:1) and human 5HT3 IPD (SEQ ID NO:4) constructs. [Figure 1D] Figure 1 shows exemplary amino acid sequences of chimeric LGICs. Mutations at amino acid residue 77 (e.g., W77F or W77Y) conferred sensitivity to granisetron and tropisetron. Mutations at amino acid residue 79 (e.g., Q79G) were most effective against several agonists. Mutations at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. Potency was significantly enhanced when LBD mutations were combined with mutations at amino acid residue 298 of the GlyR or GABAC IPD. Potency was also enhanced when α7 nAChR LBD mutations were combined with mutations at amino acid residues G175 and P216. D) Amino acid sequence of the α7-GABAc chimeric receptor (SEQ ID NO: 10) comprising the human α7 nAChR LBD (SEQ ID NO: 2) and human GABAC IPD (SEQ ID NO: 9) constructs. [Figure 1E]Figure 1 shows exemplary amino acid sequences of chimeric LGICs. Mutation of amino acid residue 77 (e.g., W77F or W77Y) conferred sensitivity to granisetron and tropisetron. Mutation of amino acid residue 79 (e.g., Q79G) was most effective against some agonists. Mutation of amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. Potency was significantly enhanced when LBD mutations were combined with mutations at amino acid residue 298 of the GlyR or GABAC IPD. Potency was also enhanced when α7 nAChR LBD mutations were combined with mutations at amino acid residues G175 and P216. E) Amino acid sequence of rat nAChR sequence (SEQ ID NO: 12). [Figure 2] Figure 2 shows the EC50 of tropisetron for the α7-5HT3 chimeric LGIC and variants of the chimeric LGIC with LBD mutations at the positions shown in Figure 1. Multiple mutations at Gln79 showed similar or improved potency compared to the unmodified α7-5HT3 channel (arrows). [Figure 3A] Figure 3 shows the relative potency of known nAChR agonists on α7-5HT3 chimeric LGICs. A) Graph of EC50 normalized to unmodified α7-5HT3 chimeric channels (logarithmic scale). *P<0.05 indicates a statistically significant change in potency (ANOVA followed by Dunn's test). [Figure 3B] Figure 3 shows the relative potency of known nAChR agonists against α7-5HT3 chimeric LGIC. B) Chemical structures of known nAChR agonists. [Figure 4A] Figure 4 shows the relative potency of known nAChR agonists on α7-GlyR chimeric LGICs. 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 against α7-GlyR chimeric LGICs. B) Graph of 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 the EC50 for α7-GlyRA298G (logarithmic scale) normalized to the unmodified α7-GlyR chimeric channel and compared to the double mutant channel α7Q79G-GlyRA298G. *P<0.05 indicates a statistically significant change in potency (ANOVA followed by Dunn's test). [Figure 5A] Figure 5 shows the schematic structures of LGIC agonists with substitution patterns most compatible with potency enhancement for α7Q79G-5HT3 and α7Q79G-GlyRA298G. A) Generalized structure showing properties associated with enhanced potency. [Figure 5B] Figure 5 shows the schematic structures of LGIC agonists with substitution patterns most compatible with potency enhancement for α7Q79G-5HT3 and α7Q79G-GlyRA298G. B) The specific pharmacophores represented in (A) are quinuclidine, tropane, and 9-azabicyclo[3.3.1]nonane core structures. [Figure 5C] Figure 5 shows the schematic structures of LGIC agonists with substitution patterns most compatible with potency enhancement for α7Q79G-5HT3 and α7Q79G-GlyRA298G. C) Exemplary synthetic molecules showing high potency for α7Q79G-GlyRA298G, α7Q79G, Y115F, G175K-GlyR, α7W77F, Q79G, G175K-GlyR. [Figure 6A]Figure 6 shows mutations that reduce the association of the chimeric LCIG α7 nAChR LBD with the unmodified LBD. A) Charge-reversal schematic potential configuration for transfection of two epitope-tagged (HA and V5) constructs encoding α7-5HT3 (top) or two constructs encoding α7-5HT3-HA and α7R21D, E41R-5HT3-V5. Here, association between the two different epitope-tagged subunits would be unfavorable due to the charge-reversal mutations at the subunit interface. [Figure 6B] Figure 6 shows mutations that reduce the association of the chimeric LCIG α7 nAChR LBD with the unmodified LBD. B) Whole-cell recordings in HEK cells expressing V5 epitope-tagged α7R21D, E41R-5HT3 show a robust response to PNU-282987. [Figure 6C] Figure 6 shows mutations that reduce the association of the chimeric LCIG α7 nAChR LBD with the unmodified LBD. C) Association of the α7-5HT3 LGIC with the HA and V5 epitope tags in HEK cells was examined by HA immunoprecipitation (left) or total lysate isolation followed by Western blotting with either anti-HA (top) or anti-V5 antibodies (bottom). In cells coexpressing the channel with the HA and V5 epitopes, anti-HA IP followed by anti-V5 immunoblotting showed coimmunoprecipitation of each type of unmodified channel, but not the charge-reversal mutations in the LBD α7R21D and E41R-5HT3-V5. The MW of α7-5HT3 is approximately 48 kD (arrow). [Figure 7] Figure 7 shows that chimeric LGICs can be regulated using exogenous ligands. Cortical neurons from mouse brains transduced with α7Q79G-GlyRA298G chimeric LGICs via adeno-associated viral (AAV) vectors fire action potentials in response to a 40 pA current injection (PRE), which is potently suppressed by 30 nM tropisetron. After washout of tropisetron (WASH), neuronal firing recovers. [Figure 8A]Figure 8 shows the activity of agonists against chimeric LGICs with the G175K mutation. A) Graph of the EC50 for the Q79G G175K LBD mutant against known agonists (logarithmic scale), normalized to the unmodified α7-GlyR chimeric channel. [Figure 8B] Figure 8 shows the activity of agonists for a chimeric LGIC with a G175K mutation. B) Graph of EC50 for ACh and tropisetron for channels with mutations in an α7-GlyR chimeric LGIC. Mutations resulting in channels with high potency for tropisetron and low potency for the endogenous ligand, acetylcholine (ACh), are optimal (gray shading). Unmod: Unmodified α7-GlyR chimeric LGIC. [Figure 8C] Figure 8 shows the activity of agonists against chimeric LGICs containing the G175K mutation. C) Action potentials of cortical neurons from mouse brains transduced with α7Q79G, Y115F, G175K-GlyR chimeric LGICs. The neurons fire in response to current injection (PRE) and are potently inhibited by 100 nM tropisetron. After washout of tropisetron (WASH), the neurons recover. [Figure 9A] Figure 9 shows the activity of agonists on chimeric LGICs with the L131G mutation. A) Graph of EC50 (logarithmic scale) for L131 LBD mutants against known agonists, 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 the activity of agonists against a chimeric LGIC with the L131G mutation. C) Graph showing that the mutation resulting in a channel with high potency for varenicline and low potency for the endogenous ligand, acetylcholine (ACh), is optimal (gray shading). Unmod: Unmodified α7-GlyR chimeric LGIC. [Figure 9D] Figure 9 shows the activity of agonists against a chimeric LGIC containing the L131G mutation. D) Action potentials of cortical neurons from mouse brains transduced with α7L131G, Q139L, Y217F-GlyR chimeric LGIC. The neurons fire in response to current injection (PRE) and are potently inhibited by 10 nM varenicline, even at over six-fold higher injected currents. After washout of tropisetron (WASH), neuronal firing recovers. [Figure 10] Figure 10 shows the chemical structures of LGIC agonists. A) Chemical structures of LGIC agonists with substitution patterns most compatible with enhanced potency for α7Q79G,Y115F,G175K-GlyR. B) Chemical structures of LGIC agonists with substitution patterns most compatible with enhanced potency for α7L131G,Q139L,Y217F-GlyR or α7L131G,Q139L,Y217F-5HT3 HC. DETAILED DESCRIPTION OF THE INVENTION

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

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

[0028] Qualified LGIC As used herein, a "modified" LGIC is an LGIC containing at least one LGIC subunit. The modified LGIC subunit may contain at least one modified amino acid (e.g., amino acid substitution) in the LBD and / or at least one modified amino acid (e.g., amino acid substitution) in the IPD. The modified LGIC subunits described herein may be modifications of LGIC from any appropriate species (e.g., human, rat, mouse, dog, cat, horse, cow, goat, pig, or monkey). In certain cases, the modified LGIC may contain 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 a homomer (e.g., one having any number of the same modified LGIC subunits) or a heteromer (e.g., one having at least one modified LGIC subunit and any number of different LGIC subunits). In certain cases, the modified LGIC described herein may be a homomeric modified LGIC. The modified LGIC described herein may contain 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 the cell membrane in response to ligand binding. For example, LGICs can conduct sodium (Na) through the cell membrane in response to ligand binding. + ), potassium (K + ), calcium (Ca 2+ ), and / or chloride (Cl - ) ions. Examples of LGICs include Cys-loop receptors (e.g., AChRs, such as nAChRs (e.g., muscle-type nAChRs or neuronal-type nAChRs)), gamma-aminobutyric acid (GABA; e.g., GABA A and GABA AThese include, but are not limited to, -ρ (also known as GABAc) receptors, GlyRs, GluCl receptors, and 5HT3 receptors), ionotropic glutamate receptors (iGluRs, e.g., AMPA receptors, kainate receptors, NMDA receptors, and delta receptors), ATP-gated channels (e.g., P2X), and phosphatidylinositol 4,5-bisphosphate (PIP2)-gated channels. When the modified LGIC described herein is a chimeric LGIC, the chimeric LGIC can include an LBD selected from any suitable LGIC and an IPD selected from any suitable LGIC. When the LGIC includes multiple different subunits (e.g., when a neuronal nAChR includes α4, β2, and α7 subunits), the LBD and / or IPD can be selected from any subunit. For example, the LBD from an nAChR can be an α7 LBD. A representative rat α7 nAChR amino acid sequence (including both the LBD and IPD) is as follows: TIFF0007755922000013.tif68158

[0031] In certain cases, the modified LGIC subunits described herein may comprise an LBD derived from the α7 nAChR. Examples of α7 nAChR LBDs include, but are not limited to, the human α7 nAChR LBD having the amino acid sequence set forth in SEQ ID NO:1, the human α7 nAChR LBD having the amino acid sequence set forth in SEQ ID NO:2, and the human α7 nAChR LBD having the amino acid sequence set forth in SEQ ID NO:11. In certain cases, the α7 nAChR LBD may be a homolog, ortholog, or paralog of the human α7 nAChR LBD set forth in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:11. In certain cases, the α7 nAChR LBD has at least 75% sequence identity (e.g., at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97%, or at least 99% sequence identity) to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:11. may have: TIFF0007755922000014.tif119158

[0032] In certain cases, the modified LGIC subunit described herein may comprise an IPD derived from the 5HT3 receptor. Examples of 5HT3 IPDs include, but are not limited to, mouse 5HT3 IPD having the amino acid sequence set forth in SEQ ID NO:3 and human 5HT3 IPD having the amino acid sequence set forth in SEQ ID NO:4. In certain cases, the 5HT3 IPD may be a homolog, ortholog, or paralog of the 5HT3 IPD set forth in SEQ ID NO:3 or SEQ ID NO:4. In certain cases, the 5HT3 IPD may have at least 75% sequence identity (e.g., at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97%, or at least 99% sequence identity) to SEQ ID NO:3 or SEQ ID NO:4. TIFF0007755922000015.tif76158

[0033] In certain cases, the modified LGIC subunit described herein may comprise an IPD from a GlyR. An example of a GlyR IPD includes, but is not limited to, a mouse GlyR IPD having the amino acid sequence set forth in SEQ ID NO:5. In certain cases, the GlyR IPD may be a homolog, ortholog, or paralog of the human GlyR IPD set forth in SEQ ID NO:5. In certain cases, the GlyR IPD may have at least 75% sequence identity (e.g., at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97%, or at least 99% sequence identity) to SEQ ID NO:5. TIFF0007755922000016.tif33158

[0034] In certain cases, the modified LGIC subunits described herein are fused to IPDs derived from GABA receptors (e.g., GABA A -ρ, also known as GABAc).A Examples of -ρ IPDs include, but are not limited to, human GABA receptor agonists having the amino acid sequence set forth in SEQ ID NO:9. A -ρ IPD. In certain cases, GABA A -ρ IPD is a human GABA A -ρ may be a homolog, ortholog, or paralog of an IPD. In certain cases, GABA A The -ρ IPD may have at least 75% sequence identity (e.g., at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97%, or at least 99% sequence identity) to SEQ ID NO:9. TIFF0007755922000017.tif32158

[0035] To calculate the percentage of sequence identity, two sequences are aligned and the number of identical amino acid residues between the two sequences is determined. The number of identical matches is divided by the length of the aligned region (i.e., the number of aligned amino acid residues) and multiplied by 100 to obtain the percentage of sequence identity. It is understood that the length of the aligned region can be a portion of one or both sequences up to the full length of the shortest sequence. It is also understood that a single sequence can be aligned with two or more other sequences and therefore have different percentage sequence identity values ​​across each aligned region. Aligning two or more sequences to determine the percentage of sequence identity can be performed using the computer program ClustalW with default parameters, which calculates the best match between a query and one or more subject sequences and aligns them so that identity, similarity, and difference can be determined. See, for example, Chenna et al., 2003, Nucleic Acids Res., 31(13):3497-500.

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

[0037] When the modified LGIC subunits described herein are chimeric LGIC subunits, the chimeric LGIC subunits can include various fusion points connecting the LBD and IPD, such that the number of amino acids in the LBD can vary when the LBD is fused to different IPDs to form chimeric channel subunits. For example, the length of the α7 nAChR LBD used to form a chimeric LGIC subunit with a 5HTS IPD is different from the length of the α7 nAChR LBD used to form a chimeric LGIC subunit with a GlyR IPD (e.g., compare Figures 1A and 1C with Figure 1B).

[0038] The modified LGIC subunits described herein can include an LBD with at least one modified amino acid and / or an IPD with at least one modified amino acid. For example, the modified LGIC subunits described herein can include an α7 LBD with at least 75% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 and an amino acid substitution at amino acid residues 27, 41, 77, 79, 131, 139, 141, 175, 210, 216, 217, and / or 219. For example, the modified LGIC subunits described herein can include a GlyR IPD with at least 75% sequence identity to the sequence set forth in SEQ ID NO:5 and an amino acid substitution at amino acid residue 298 of an α7-GlyR chimeric receptor (e.g., SEQ ID NO:7). For example, the modified LGIC subunit described herein may comprise a GABAC IPD having at least 75% sequence identity to SEQ ID NO:9 and an amino acid substitution at amino acid residue 298 of an α7-GABAc chimeric receptor (e.g., SEQ ID NO:10). In certain cases, the modified LGIC subunit described herein may contain more than one amino acid modification (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more). The modifications may be amino acid substitutions. In certain cases, the modified amino acid may confer pharmacological selectivity to the modified LGIC. For example, the modified amino acid may confer selective binding of an exogenous LGIC ligand to the modified LGIC. For example, the modified amino acid may result in reduced (minimized or eliminated) binding of an unmodified LGIC subunit (an LGIC subunit lacking the modification and / or an endogenous LGIC subunit) to the modified LGIC. For example, the modified amino acid may confer reduced (minimized or eliminated) binding of an endogenous LGIC ligand to the modified LGIC.

[0039] In certain embodiments, the modified LGIC subunits described herein may contain at least one modified amino acid that confers selective binding to an exogenous LGIC ligand (e.g., enhanced binding or enhanced efficacy) to the modified LGIC. Binding to the exogenous LGIC ligand may be more selective than binding to the endogenous LGIC ligand. A modified LGIC subunit with selective binding to an exogenous LGIC ligand may comprise any suitable LBD (e.g., α7 LBD). In certain embodiments, the modified LGIC subunit may comprise the α7 LBD set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12, and the amino acid modification may be a substitution at residues 77, 79, 131, 139, 141, 175, and / or 216. In certain cases, the tryptophan at amino acid residue 77 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with a hydrophobic amino acid residue, such as phenylalanine (e.g., W77F), tyrosine (e.g., W77Y), or methionine (e.g., W77M). For example, a modified LGIC subunit described herein can include an α7 LBD set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12, and having a W77F substitution. In certain cases, the glutamine at amino acid residue 79 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with an amino acid residue such as alanine (e.g., Q79A), glycine (e.g., Q79G), or serine (e.g., Q79S). For example, a modified LGIC subunit described herein can include an α7 LBD with a Q79G substitution. In certain cases, the leucine at amino acid residue 131 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with an amino acid residue such as alanine (e.g., L131A), glycine (e.g., L131G), methionine (e.g., L131M), asparagine (e.g., L131N), glutamine (e.g., L131Q), valine (e.g., L131V), or phenylalanine (e.g., L131F).In certain cases, the glycine at amino acid residue 175 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with an amino acid residue such as lysine (e.g., G175K), alanine (e.g., G175A), phenylalanine (e.g., G175F), histidine (e.g., G175H), methionine (e.g., G175M), arginine (e.g., G175R), serine (e.g., G175S), or valine (e.g., G175V). In certain cases, the proline at amino acid residue 216 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with an amino acid residue such as isoleucine (e.g., P216I). Modified LGIC subunits having selective binding to exogenous LGIC ligands can be substituted with any suitable IPD (e.g., GlyR IPD or GABA). A In certain embodiments, the modified LGIC subunit may comprise a GlyR IPD set forth in SEQ ID NO:5, where the amino acid modification may be a substitution at amino acid residue 298 of an α7-GlyR chimeric receptor (e.g., SEQ ID NO:7). In certain cases, the alanine at amino acid residue 298 of SEQ ID NO:7 can be substituted with an amino acid residue such as glycine (e.g., A298G). In certain embodiments, the modified LGIC subunit may comprise a GABA IPD set forth in SEQ ID NO:9. A -ρIPD, the amino acid modification of which may be α7-GABA A The substitution may be at amino acid residue 298 of the -ρ chimeric receptor (e.g., SEQ ID NO: 10). In certain cases, the tryptophan at amino acid residue 298 of SEQ ID NO: 10 can be substituted with an amino acid residue such as alanine (e.g., W298A).

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

[0042] In certain embodiments, the modified LGIC subunits described herein may contain at least one modified amino acid that confers reduced (e.g., minimized or eliminated) binding to the modified LGIC with unmodified LGIC subunits. Binding to modified LGIC subunits with the same modification may be more selective than binding to unmodified LGIC subunits. The unmodified LGIC subunit may be an LGIC subunit lacking a modification that confers reduced binding to the modified LGIC with unmodified LGIC subunits, or the unmodified LGIC may be an endogenous LGIC subunit. The modification that confers reduced binding to the modified LGIC with unmodified LGIC subunits may be a charge-reversal modification. The modified LGIC subunit with reduced binding to unmodified LGIC subunits may comprise any suitable LBD (e.g., α7 LBD). In certain embodiments, the modified LGIC subunit may comprise the α7 LBD set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12, and the amino acid modification may be a substitution at amino acid residues 27 and / or 41. For example, the arginine at amino acid residue 27 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 may be substituted with aspartic acid (e.g., R27D). For example, the glutamic acid at amino acid residue 41 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with arginine (e.g., E41R). In certain cases, the modified LGIC subunits described herein can include an α7 LBD with an R27D substitution and E41R.

[0043] In certain embodiments, the modified LGIC subunits described herein may contain at least one modified amino acid that confers reduced (e.g., minimized or eliminated) binding of an endogenous LGIC ligand to the modified LGIC. The endogenous LGIC ligand may be ACh. The modified LGIC subunit with reduced binding of an endogenous LGIC ligand may comprise any appropriate IPD (e.g., GlyR LBD). For example, the modified LGIC subunit may comprise the α7 LBD set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12, and the amino acid modification may be a substitution at amino acid residues 115, 131, 139, 210, 217, and / or 219. In certain cases, the tyrosine at amino acid residue 115 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 may be substituted with phenylalanine (e.g., Y115F). In certain cases, the leucine at amino acid residue 131 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with an amino acid residue such as alanine (e.g., L131A), glycine (e.g., L131G), methionine (e.g., L131M), asparagine (e.g., L131N), glutamine (e.g., L131Q), valine (e.g., L131V), or phenylalanine (e.g., L131F). In certain cases, the glutamine at amino acid residue 139 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with glycine (e.g., Q139G) or leucine (e.g., Q139L). In certain cases, the tyrosine at amino acid residue 210 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with phenylalanine (e.g., Y210F). In certain cases, the tyrosine at amino acid residue 217 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with phenylalanine (e.g., Y217F). In certain cases, the aspartic acid at amino acid residue 219 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO:12 can be substituted with alanine (e.g., D219A).

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

[0045] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-5HT3 LGIC subunit (SEQ ID NO: 6) having a human α7 nAChR LBD (SEQ ID NO: 1) with Q79G and Y115F amino acid substitutions, and a mouse 5HT3 IPD (SEQ ID NO: 3).

[0046] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-5HT3 LGIC subunit (SEQ ID NO: 6) having a human α7 nAChR LBD (SEQ ID NO: 1) with a Q79G amino acid substitution and a Q139G amino acid substitution, and a mouse 5HT3 IPD (SEQ ID NO: 3).

[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) with a Q79G and a Y115F amino acid substitution, and a human GlyR IPD (SEQ ID NO: 5) with 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) with a Q79G and Q139G amino acid substitution, and a human GlyR IPD (SEQ ID NO: 5) with 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) with an R27D, E41R, Q79G, and Y115F amino acid substitution, and a human GlyR IPD (SEQ ID NO: 5) with an A298G amino acid substitution.

[0050] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with a substitution at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N), and a human GlyR IPD (SEQ ID NO: 5).

[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) with 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 comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with a substitution at amino acid residue 175 (e.g., G175K) and a human GlyR IPD (SEQ ID NO: 5).

[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) with substitutions at amino acid residues 131 (e.g., L131G, L131A, L131M, or L131N) and 139 (e.g., Q139L), and a human 5HT3 IPD (SEQ ID NO: 4) with R420Q, R424D, and R428A substitutions.

[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) with substitutions at amino acid residues 131 (e.g., L131G, L131A, L131M, or L131N), 139 (e.g., Q139L), and 217 (e.g., Y217F), and a human 5HT3 IPD (SEQ ID NO: 4) with R420Q, R424D, and R428A substitutions.

[0059] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions at amino acid residues 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 comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions at amino acid residues 175 (e.g., G175K), 115 (e.g., Y115F), and 79 (e.g., Q79G), and a human GlyR IPD (SEQ ID NO: 5).

[0061] In certain cases, the modified LGIC described herein may comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with substitutions at amino acid residues 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 comprise at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) with a substitution at amino acid residue 216 (e.g., P216I), and a human GlyR IPD (SEQ ID NO: 5).

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

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

[0067] The present specification also provides nucleic acids encoding the modified LGIC subunits described herein, as well as constructs for expressing nucleic acids encoding the modified LGIC subunits described herein (e.g., plasmids, non-viral vectors, viral vectors (e.g., adeno-associated virus, herpes simplex virus, or lentiviral vectors, etc.)). The nucleic acids encoding the 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., neuron-specific, muscle-specific, glial-specific), and neural subtype-specific). Examples of promoters that can be used to drive expression of nucleic acids encoding the 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 the modified LGIC subunits described herein can be operably linked to a neuron-specific promoter.

[0068] The present specification also provides cells (e.g., mammalian cells) having the modified LGIC described herein. Mammalian cells having the modified LGIC described herein can be obtained by any suitable method. In certain cases, a pre-assembled modified LGIC can be provided to the cell. In certain cases, a nucleic acid encoding a modified LGIC subunit described herein can be provided to the cell under conditions in which the modified LGIC subunit is translated and under conditions in which multiple (e.g., 3, 4, 5, 6, or more) modified LGIC subunits can be assembled into the modified LGIC described herein.

[0069] LGIC Ligand The present specification also provides LGIC ligands that can bind to and activate the modified LGIC described herein. LGIC ligands that can bind to and activate the modified LGIC described herein can be exogenous or endogenous. LGIC ligands that can bind to and activate the modified LGIC described herein can be naturally occurring or synthetic. LGIC ligands that can bind to and activate the modified LGIC described herein can be canonical or non-canonical. LGIC ligands that can bind to and activate the modified LGIC described herein can be agonists or antagonists. In certain cases, the LGIC ligand is an exogenous LGIC agonist. Examples of LGIC ligands include, but are not limited to, ACh, nicotine, epivastatin, cytisine, RS56812, tropisetron, nortopisetron, PNU-282987, PHA-543613, Compound 0353, Compound 0354, Compound 0436, Compound 0676, Compound 702, Compound 723, Compound 725, granisetron, ivermectin, mequitazine, promazine, varenicline, Compound 765, Compound 770, 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-dioxide, Compound 773, and Compound 774 (see, e.g., Figures 3B, 5C, 10A, and 10B).

[0070] An LGIC ligand that can bind to and activate a modified LGIC described herein may have selective binding (e.g., enhanced binding or increased potency) for the modified LGIC described herein. In certain cases, an LGIC ligand that can bind to and activate a modified LGIC described herein does not bind to and activate an endogenous receptor. An LGIC ligand that selectively binds to and activates a modified LGIC described herein over an unmodified LGIC ligand (e.g., a modified LGIC having at least one amino acid modification that confers pharmacological selectivity to the modified LGIC) may be described as having enhanced potency for the modified LGIC. In certain cases, a modified LGIC subunit described herein that selectively binds to an exogenous LGIC ligand may have an enhanced potency of at least 5-fold (e.g., at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at least 95-fold, at least 100-fold, at least 125-fold, at least 150-fold, at least 200-fold, at least 250-fold, or at least 300-fold) relative to the modified LGIC. For example, an LGIC ligand that selectively binds to and activates a modified LGIC may have enhanced potency relative to the modified LGIC by about 10 to about 300 times (e.g., about 10 to about 250 times, about 10 to about 200 times, about 10 to about 150 times, about 10 to about 100 times, about 25 to about 300 times, about 50 to about 300 times, about 100 to about 300 times, about 200 to about 300 times, about 25 to about 250 times, about 50 to about 200 times, or about 100 to about 150 times). In certain cases, an LGIC ligand that binds to and activates a modified LGIC described herein may have a ligand potency of less than 25 nM (e.g., less than 22 nM, less than 20 nM, less than 17 nM, less than 15 nM, less than 13 nM, less than 12 nM, less than 11 nM, less than 10 nM, less than 5 nM, less than 2 nM, or less than 1 nM).For example, an LGIC ligand that binds to and activates a modified LGIC described herein may have a ligand potency of less than 15 nM. In certain cases, an LGIC ligand may have an EC50 of less than 25 nM (e.g., less than 22 nM, less than 20 nM, less than 17 nM, less than 15 nM, less than 13 nM, less than 12 nM, less than 11 nM, or less than 10 nM) for a modified LGIC subunit described herein. For example, an LGIC ligand (e.g., tropisetron) may bind to a modified LGIC subunit (e.g., α7) described herein. Q79G -GlyR A298G For example, an LGIC ligand (e.g., nortropisetron) may have an EC50 of about 11 nM for a modified LGIC subunit (e.g., α7) described herein. Q79G、Y115F -GlyR A298G ) of about 13 nM. In certain cases, an LGIC ligand may have an EC50 of greater than 20 μM (e.g., greater than 22 μM, greater than 25 μM, greater than 35 μM, greater than 50 μM, greater than 65 μM, greater than 80 μM, or greater than 100 μM) for a modified LGIC subunit described herein. For example, an LGIC ligand (e.g., ACh) may have an EC50 of greater than 20 μM (e.g., greater than 22 μM, greater than 25 μM, greater than 35 μM, greater than 50 μM, greater than 65 μM, greater than 80 μM, or greater than 100 μM) for a modified LGIC subunit described herein (e.g., α7 Q79G、Y115F -GlyR A298G ) may have an EC50 of greater than 100 μM.

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

[0072] LGIC ligands capable of binding to and activating the modified LGICs described herein have the formula I: TIFF0007755922000018.tif32128 (wherein X1 and X2 can independently be CH, CH2, O, NH, or NMe; each n can independently be 0 or 1; Y can be O or S; A can be an aromatic substituent; and R can be H or pyridinylmethylene). Examples of aromatic substituents include, but are not limited to, 4-chloro-benzene, 1H-indole, 4-(trifluoromethyl)benzene, 4-chlorobenzene, 2,5-dimethoxybenzene, 4-chloroaniline, aniline, 5-(trifluoromethyl)pyridin-2-yl, 6-(trifluoromethyl)nicotine, and 4-chloro-benzene.

[0073] An LGIC ligand capable of binding to and activating the modified LGIC described herein can be quinuclidine. Quinuclidine has the formula II: TIFF0007755922000019.tif18128 (wherein X3 can be O, NH, or CH2; Y can be O or S; A can be an aromatic substituent; and R can be H or pyridinylmethylene) The aromatic substituent may have the structure: Examples of aromatic substituents include, but are not limited to, 1H-indole, 4-(trifluoromethyl)benzene, 4-chlorobenzene, 2,5-dimethoxybenzene, 4-(trifluoromethyl)benzene, 4-chloroaniline, aniline, 5-(trifluoromethyl)pyridin-2-yl, 6-(trifluoromethyl)nicotine, 3-chloro-4-fluorobenzene, 4-chloro-benzene, and 1H-indole. Examples of quinuclidines include, but are not limited to, compounds PNU-282987, PHA-543613, 0456, 0434, 0436, 0354, 0353, 0295, 0296, and 0676 (see, for example, Figure 5C, Table 3, and Table 6).

[0074] An LGIC ligand capable of binding to and activating a modified LGIC described herein can be a tropane. The tropane has formula III: TIFF0007755922000020.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] An LGIC ligand capable of binding to and activating the modified LGIC described herein can be 9-azabicyclo[3.3.1]nonane, which has formula IV: TIFF0007755922000021.tif27128 (wherein X1 can be CH, X2 can be NH or NMe, X3 can be O, NH, or CH; Y can be O or S; A can be an aromatic substituent) The aromatic substituent may have the structure: An example, but is not limited to, 4-chloro-benzene. Examples of 9-azabicyclo[3.3.1]nonanes include, but are not limited to, Compound 0536, Compound 0749, Compound 0751, Compound 0760, and Compound 0763 (see, for example, Figure 5C, Table 3, and Table 6).

[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: TIFF0007755922000022.tif13128 (wherein R is H or CH3 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., Figure 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: TIFF0007755922000023.tif25128 (in the formula, R=H, F, NO2) Examples of 1,4-diazabicyclo[3.2.2]nonanes include, but are not limited to, 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-dioxide, compound 0773, and compound 0774 (see, e.g., Figure 10B, Table 6, and Table 9).

[0078] How to use Also provided herein are methods using the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein. The LGIC ligands capable of binding to and activating the modified LGICs can be used to activate the modified LGICs with temporal and / or spatial control based on delivery of the ligand.

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

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

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

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

[0085] When the materials and methods described herein are used to treat mammals with channelopathies and / or mammals at risk of developing channelopathies, the channelopathies can be any channelopathies. As used herein, a channelopathies can be any disease or disorder that is caused by abnormal ion channel function and / or abnormal ligand function, or that can be alleviated by regulated ion channel function and / or altered cellular ion flux (e.g., calcium ion flux). A channelopathies can be congenital or acquired. Examples of channelopathies include, but are not limited to, Bartter syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT), congenital hyperinsulinemia, cystic fibrosis, Dravet syndrome, transient ataxia, erythromelalgia, generalized epilepsy (e.g., with febrile seizures), familial hemiplegic migraine, fibromyalgia, hyperkalemic periodic paralysis, hypokalemic periodic paralysis, Lambert-Eaton myasthenic syndrome, long QT syndrome (e.g., Romano-Ward syndrome), short QT syndrome, malignant hyperthermia, mucolipidosis type IV, myasthenia gravis, myotonia congenita, neuromyelitis optica, neuromyotonia, nonsyndromic hearing loss, congenital paramyotonia, retinitis pigmentosa, Timothy syndrome, tinnitus, seizures, trigeminal neuralgia, and multiple sclerosis. Alternatively, or in addition, the materials and methods described herein can be used for other applications, including, but not limited to, pain treatment, cancer cell treatment, appetite control, spasticity treatment, muscular dystonia treatment, tremor treatment, and movement disorder treatment.

[0086] In certain cases, the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein can be used to regulate cellular activity. The cellular activity regulated using the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein can be any cellular activity. Examples of cellular activity include, but are not limited to, active transport (e.g., ion transport), passive transport, excitation, inhibition, ion flux (e.g., calcium ion flux), and exocytosis. Cellular activity can be increased or decreased. For example, the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein can be used to regulate (e.g., increase) ion transport across a cell's membrane. For example, the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein can be used to regulate (e.g., increase) cellular excitability.

[0087] The modified LGICs described herein and LGIC ligands that can bind to and activate the modified LGICs described herein can be used to regulate the activity of any type of cell in a mammal. The cell can be a neuron, a glial cell, a muscle cell, an immune cell (e.g., neutrophils, eosinophils, basophils, lymphocytes, and monocytes), an endocrine cell, or a stem cell (e.g., an embryonic stem cell). In certain cases, the cell can be an excitable cell. The cell can be in vivo or ex vivo.

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

[0089] The modified LGIC described herein can be detected by any suitable method (e.g., to confirm its presence in cells). In certain cases, an agent that selectively binds the modified LGIC can be used to detect the modified LGIC. Examples of agents that can be used to bind to the modified LGIC described herein include, but are not limited to, antibodies, proteins (e.g., bungarotoxin), and small molecule ligands (e.g., PET ligands). The agent that selectively binds to the modified LGIC can include a detectable label (e.g., a fluorescent label, a radioactive label, a positron-emitting label, and an enzyme label). Methods for detecting LGIC expression in cells can include fluorescence imaging, autoradiography, functional MRI, PET, and SPECT.

[0090] The modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein can be administered to a mammal having a channelopathy and / or at risk of developing a channelopathy as a combination therapy with one or more additional agents / therapies used to treat the channelopathy. For example, a combination therapy used to treat a mammal having a channelopathy as described herein can include administering a modified LGIC described herein and an LGIC ligand capable of binding to and activating the modified LGIC described herein, and treating with acetazolamide, dichlorophenamide, mexilitine, glucose, calcium gluconate, L-DOPA, muscle stimulation, spinal cord stimulation, brain stimulation, and / or neurostimulation.

[0091] In embodiments in which the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein are used in combination with additional agents / therapies used to treat channelopathies, the one or more additional agents can be administered simultaneously or independently. For example, the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein can be administered first, followed by the one or more additional agents, or vice versa. In embodiments in which the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein are used in combination with one or more additional therapies used to treat channelopathies, the one or more additional therapies can be administered simultaneously or independently with the administration of the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein. For example, the modified LGICs described herein and LGIC ligands capable of binding to and activating the modified LGICs described herein can be administered before, during, or after the administration of the one or more additional therapies.

[0092] In certain cases, the modified LGICs described herein and / or LGIC ligands capable of binding to and activating the modified LGICs described herein can be formulated into pharmaceutically acceptable compositions for administration to mammals having or at risk of developing a channelopathy. For example, a therapeutically effective amount of the modified LGICs described herein (e.g., nucleic acids encoding the modified LGICs described herein) and / or LGIC ligands capable of binding to and activating the modified LGICs described herein can be formulated together with one or more pharmaceutically acceptable carriers (excipients) and / or diluents. Pharmaceutical compositions can be formulated for administration in solid or liquid forms, including, but not limited to, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules.

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

[0095] A pharmaceutically acceptable composition comprising a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and / or an LGIC ligand capable of binding to and activating a modified LGIC described herein can be administered locally or systemically. In certain cases, a composition comprising a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and / or an LGIC ligand capable of binding to and activating a modified LGIC described herein can be administered systemically to a mammal (e.g., a human) by intravenous or oral administration, or by inhalation. In certain cases, a composition comprising a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and / or an LGIC ligand capable of binding to and activating a modified LGIC described herein can be administered locally to a target tissue of a mammal (e.g., a human) by transdermal, subcutaneous, intramuscular, intracranial, or open surgical administration (e.g., injection).

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

[0098] The effective period for administering a therapeutically effective amount of a composition comprising a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and / or a LGIC ligand capable of binding to and activating a modified LGIC described herein is any period that ameliorates symptoms of a channelopathy without causing significant toxicity to a mammal. For example, the effective period can vary from several days to several weeks, several months, or even several years. In certain cases, the effective period for treating a channelopathy can range from about one month to about 10 years. Multiple factors influence the actual effective period used for a particular treatment. For example, the effective period can vary depending on the frequency of administration, the effective amount, the use of multiple therapeutic agents, the route of administration, and the severity of the channelopathy being treated.

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

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

[0101] Example 1: Potency-enhancing ligand-binding domain mutations A panel of 41 α7-5HT3 chimeric channels with mutated LBDs was screened against a panel of 51 clinically used drugs with chemical similarity to nicotinic receptor agonists. The mutations are at the residues highlighted in Figure 1. The screen identified a Gln in the α7 nAChR LBD that enhances potency against the known nAChR agonist tropisetron. 79 We identified mutations in the α7 nAChR (Figure 2). These mutations (Q79A, Q79G, Q79S) reduce the size of the amino acid side chain. Several mutant ion channel-ligand combinations provided up to a 12-fold improvement in potency (Table 1, Figure 3). Standard α7 nAChR agonists, ACh, nicotine, epibatidine, and the smoking cessation drug varenicline were not significantly affected by the Q79A, Q79G, or Q79S mutations. However, a subset of α7 nAChR agonists showed enhanced potency with some mutations. Cytisine, RS56812, tropisetron, nortropisetron, and PNU-282987 all exhibited potency enhancement with the α7 nAChR. Q79G Furthermore, nortropisetron and PNU-282987 each demonstrated significantly improved efficacy against α7 Q79A -5HT3 and α7 Q79Sshowed significantly enhanced potency for α7 agonists. In general, agonists based on quinuclidine or tropane pharmacophores with linked aromatic structures that interact with the complementary binding surface of the ligand-binding domain showed improved potency with Gln79 substitution with smaller amino acid residues Ala, Gly, or Ser. For most agonists, the α7 Q79G -5HT3 was the most preferred mutant chimeric lion channel.

[0102] Table 1. Potency of nAChR agonists against chimeric cation channels mutated at Gln79 in HEK cells. Mean EC50, SEM (μM) in parentheses. TIFF0007755922000024.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-GlyR chimeric channels. Q79G Similar to the LBD mutations for α7-5HT3, nortropisetron showed significantly enhanced potency against α7-GlyR. Q79A -GlyR, and PNU-282987 has significantly enhanced potency against α7 Q79S For most agonists, the potency at α7-GlyR was significantly enhanced. Q79G -GlyR was the most preferred mutant chimeric lion channel.

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

[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 substantially ligand-free open probability, making them unsuitable for ligand-controlled manipulation of cells. α7-GlyR was effective in enhancing ivermectin efficacy at the full-length glycine receptor. A298G Mutations at α7-GlyR resulted in modest changes in the open probability in the absence of ligand. Therefore, this channel was tested for activity against a panel of known agonists. α7-GlyR activity was measured for the standard agonists ACh, nicotine, and epibatidine, as well as for varenicline and tropisetron. A298G Agonist potency was not significantly enhanced in α7 nAChR agonists. A subset of α7 nAChR agonists showed modest, up to 4-fold increases in potency: RS56812, cytisine, PNU-282987, and nortropisetron were significantly more potent. Thus, the effect of the IPD A298G mutation improved ligand potency, but, depending on the ligand structure, was not as effective as mutations in the LBD.

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

[0108] Table 2: Potency of nAChR agonists against mutant chimeric chloride channels. Mean EC 50 and SEM (μM) for agonist activity in HEK cells expressing chimeric channels. TIFF0007755922000025.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. Additionally, we also tested the known α7 nAChR agonist PHA-543613 (Walker et al. 2006, Wishka et al. 2006). Q79G -GlyR A298G These molecules generally exhibited 10- to 100-fold enhanced potency (Table 3), suggesting that for these pharmacophores, a specific range of structural features contributes to the α7 Q79G -GlyR A298G This demonstrates that the efficacy of the steroids is consistent with improved efficacy.

[0111] These results indicate that the modified LGIC 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. TIFF0007755922000026.tif233125

[0113] Example 4: Mutations that reduce acetylcholine responsiveness The α7 nAChR is relatively insensitive to ACh compared with other nAChR isoforms, and potency-enhancing mutations for tropane and quinuclidine ligands did not substantially alter the potency of acetylcholine at these channels. Therefore, the chimeric channels were further modified to reduce the acetylcholine responsiveness of these channels. Additional LBD mutations, Y115F and Q139G, significantly reduced acetylcholine responsiveness to >100 μM in certain cases, and the α7 Q79G、Y115F -5HT3, α7 Q79G、Q139G -5HT3, α7 Q79G、Q139G -GlyR A298, α7 Q79G、Y115F -GlyR A298G For example, α7 only modestly reduced the potency of certain agonists. Q79G、Y115F -GlyR A298G has an EC50 of 13 nM for nortropisetron and over 100 μM for ACh (Table 4).

[0114] Table 4: Potency of nAChR agonists against mutant chimeric chloride channels with reduced acetylcholine responsiveness. Mean EC 50 and SEM (μM) for activity in HEK cells expressing the chimeric channels. TIFF0007755922000027.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 interactions between the LBDs (Celie et al., 2004 Neuron 41: 907-14). To minimize unwanted association with endogenous α7 nAChR subunits and / or undesired association of chimeric channels, we identified potential intersubunit bridges by examining the crystal structure of an acetylcholine-binding protein (ABP) and identifying nearby intersubunit residues with opposite charges that also share homologous ionizable amino acids in the α7 nAChR receptor LBD. Charge-reversal mutations (switching the acidic member of a potential salt bridge to a basic residue and its basic partner to an acidic residue) were designed to disrupt intersubunit interactions with unmodified subunits but preserve interactions between subunits with charge-reversal mutations (Figure 6A). Chimeric LGIC subunits with charge-reversal mutations were able to selectively associate with each other without interacting with unmodified channels, e.g., endogenous α7 nAChR. The R27D, E41R double mutation in the α7 nAChR LBD resulted in functional channels (Fig. 6B). Co-expression of these charge-reversal channels with α7-5HT3 channels containing the unmodified sequence demonstrated that the charge-reversal subunit did not co-immunoprecipitate with the unmodified channel (Fig. 6C). R27D、E41R、Q79G、Y115F -GlyR A298G Combining potency-enhancing mutations and acetylcholine-blocking mutations to obtain α- and β-agonists revealed that some agonists retained high potency relative to their cognate agonists (Table 4, right column).

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

[0119] (Table 5) Agonist potency enhancement by G175K and P216I mutations in a7GlyR chimeric channels. TIFF0007755922000028.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 observed for tropisetron, granisetron, nortropisetron, PNU-282987, and PHA-543613, with significantly reduced potency for varenicline and cytisine (Table 5). G175K、Y210F For -GlyR, potency for most agonists was significantly reduced, but enhanced potency for granisetron was observed (Table 5).

[0121] To develop a channel with reduced ACh responsiveness but high potency to other agonists, we investigated the α7 G175K The -GlyR was combined with additional mutations that increased the potency of certain agonists. Combination with W77F decreased ACh potency and α7 W77F、G175K The α7-GlyR showed increased potency over the α7-GlyR for granisetron, nortopisetron, and tropisetron, but not for PNU282-987, varenicline, cytisine, or PHA-543613 (Table 5). Combining G175K with Q79G reduced ACh potency and increased α7 Q79G、G175K The α7-GlyR showed increased potency for nortropisetron, PHA-543613, and tropisetron (Table 5). However, this increased potency was not observed for other agonists, such as PNU282-987 or varenicline. G175K、Q139L -GlyR decreased ACh potency and increased potency for nortropisetron and tropisetron (Table 5).

[0122] By incorporating mutations at W77F, Q79G, L141F, Y115F, G175K, and Y210F in various combinations, further reductions in ACh potency were achieved while maintaining high potency for synthetic agonists, including those based on the tropane and quinuclidine core structures. Q79G、Y115F、G175K -GlyR reduced ACh responsiveness while maintaining a strong response to tropisetron (Table 5). These mutations also Y115F、G175KResponses to other tropane and quinuclidine core structures, particularly quinuclidine thioureas 702 and 703, and tropane esters 723, 725, 726, 736, 737, 738, and 745, were also enhanced relative to α7-GlyR and relative to α7-5HT3 (representative of endogenous α7 nAChR activity) (Table 6). Q79G、Y115F、G175K -GlyR also showed high sensitivity to ivermectin (Table 5). W77F、Q79G、G175K α7-GlyR reduced ACh responsiveness while maintaining high potency responses to tropisetron and nortropisetron (Table 5). W77F、Q79G、G175K α7-GlyR also showed enhanced potency for additional tropane-based core structures, such as compounds 723 and 725, and the clinically used drugs mequitazine and promazine (Table 6). W77F、G175K、Y210F α7-GlyR reduced ACh responsiveness but significantly improved efficacy to granisetron (Table 5). L141F、Y115F、G175K α7-GlyR reduced ACh responsiveness while conferring sensitivity to granisetron (Table 5). Q79G、Q139L、G175K -GlyR reduced ACh responsiveness but exhibited a potent response to nortropisetron (Table 5).

[0123] Table 6. Potency enhancement of tropane, quinuclidine agonist, 9-azabicyclo[3.3.1]nonane agonist, diazabicyclo[3.2.2]nonane agonist, and promazine by G175K and P216I α7GlyR chimeric channels. Indole and indazole aromatic (A) substituents attached at the 3-position. TIFF0007755922000029.tif235138TIFF0007755922000030.tif235148nd=Not determined;Bracket:SEM

[0124] α7 G175K -GlyR and α7 P216Iα7-GlyR was compatible with the non-associating mutations R27D, E41R, and GlyR IPD mutation A298G, along with mutations at Q79G, Y115F, and G175K, which further enhanced ligand potency for granisetron, epibatidine, varenicline, cytisine, PNU-282987, tropisetron, nortropisetron, and PHA-543613 (Table 7). R27D、E41R、Q79G、Y115F、G175K Combination with non-associating mutations to form 702, 723, 725, and 726 further improved potency for 702, 723, 725, and 726, with low ACh responsiveness (Table 6).

[0125] Table 7. G175K and A298G mutations in α7GlyR chimeric channels and α7GABAc (GABA A Enhanced agonist efficacy by W298A at the -ρ (also known as -ρ) channel. TIFF0007755922000031.tif79157nd=Not determined;Bracket:SEM

[0126] α7 Y115F Gly in the α7 nAChR LBD in the α7-GlyR chimeric channel 175 Further amino acid substitutions at α7 also enhanced agonist potency. Y115F The potency of tropisetron in the -GlyR chimeric channel was enhanced with additional mutations, including G175A (7.1-fold), G175F (2-fold), G175H (2.3-fold), G175K (5.6-fold), G175M (2.6-fold), G175R (5.8-fold), G175S (9.3-fold), and G175V (16.7-fold).

[0127] (Table 8) Enhanced agonist potency by G175 mutation in α7GlyR Y115F chimeric channels. TIFF0007755922000032.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 α7-GlyR reduced ACh responsiveness (6-fold) and enhanced potency to varenicline (8-fold and 17-fold, respectively) and tropisetron (2.5-fold and 3.6-fold, respectively) (Table 9). L131G -5HT3 HC reduced ACh responsiveness (5-fold) and enhanced efficacy to varenicline (16-fold) and tropisetron (2.3-fold) (Figure 9A and Table 9). L131G、Q139L -GlyR and α7 L131G、Y217F α7-GlyRs showed a similar potency enhancement for varenicline relative to α7-GlyRs (21-fold), but also decreased ACh sensitivity (−11-fold and −13-fold, respectively). Q79S、L131G α7-GlyR further improved efficacy over α7-GlyR for varenicline (89-fold) and tropisetron (15-fold). L131G、Q139L、Y217F α7-GlyR showed the greatest improvement in potency over α7-GlyR for varenicline (387-fold) and also showed a decrease in ACh potency (13-fold) (Figure 9B and Table 9). L131G、Q139L、Y217F The α7-GlyR also showed extremely high potency for Compound 770 (0.001 μM), Compound 773 (0.00034 μM), and Compound 774 (0.00013 μM) (FIG. 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 against tropisetron, nortropisetron, PHA-543613, and granisetron (Table 9). L131F-GlyR was found to substantially reduce ACh potency but did not improve potency for other agonists (Table 8). L131G -GABAc substantially reduced ACh potency but did not improve potency for other agonists (Table 9). L131G、Q139L、Y217F α7-5HT3 HC (Table 9) improved the efficacy of varenicline 131-fold over α7-5HT3 (Table 1). L131G、Q139L、Y217F -5HT3 HC also showed high potency for Compound 770 (0.007 μM), Compound 773 (0.002 μM), and Compound 774 (0.004 μM) (Table 8).

[0129] Table 9. Agonist potency enhancement by chimeric channels with the L131 mutation. TIFF0007755922000033.tif235122nd=Not determined;Bracket:SEM

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

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

[0132] These results indicate that modulated 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 offer an attractive strategy for combining drug therapy with gene therapy. This is because the use of exogenously delivered ion channels selectively coupled to drug administration allows the same ion channel and ligand to be used to modulate cellular function in a consistent manner across different cell types in a variety of indications. The identification of ion channels that are well tolerated and gated by clinically used drugs is particularly attractive for potentially extending chemical genetics to human therapeutic applications.

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

[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.

Claims

1. 1. A modified ligand-gated ion channel (LGIC) comprising at least one modified LGIC subunit, the modified LGIC subunit comprising: an alpha 7 nicotinic acetylcholine receptor (α7-nAChR) ligand binding domain (LBD) comprising amino acid substitutions selected from the group consisting of: i) an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution; ii) an L131M amino acid substitution and a Y115F amino acid substitution; iii) a W77F amino acid substitution, a Q79G amino acid substitution, and a G175K amino acid substitution; iv) a Q79G amino acid substitution, a Y115F amino acid substitution, and a G175K amino acid substitution; v) a Y115F amino acid substitution and a G175K amino acid substitution; and vi) a Q79G amino acid substitution and a P216I amino acid substitution in a sequence set forth in residues 23-224 of SEQ ID NO: 1, residues 23-229 of SEQ ID NO: 2, or residues 23-233 of SEQ ID NO: 11, numbered relative to SEQ ID NO: 1; an ion pore domain (IPD) selected from the group consisting of a serotonin 3 receptor (5HT3) IPD, a glycine receptor (GlyR) IPD, and a gamma-aminobutyric acid (GABA) receptor IPD; wherein an exogenous LGIC ligand activates the modified LGIC.

2. The modified LGIC of claim 1 , which has reduced potency to activation by endogenous acetylcholine compared to an LGIC lacking the modified LGIC subunit.

3. 3. The modified LGIC of claim 1 or 2, wherein the α7-nAChR LBD comprises an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution.

4. The modified LGIC of claim 1 or 2, wherein the α7-nAChR LBD comprises an L131M amino acid substitution and a Y115F amino acid substitution.

5. 3. The modified LGIC of claim 1 or 2, wherein the α7-nAChR LBD comprises a W77F amino acid substitution, a Q79G amino acid substitution, and a G175K amino acid substitution.

6. 3. The modified LGIC of claim 1 or 2, wherein the α7-nAChR LBD comprises a Q79G amino acid substitution, a Y115F amino acid substitution, and a G175K amino acid substitution.

7. The modified LGIC of claim 1 or 2, wherein the α7-nAChR LBD comprises a Y115F amino acid substitution and a G175K amino acid substitution.

8. The modified LGIC of claim 1 or 2, wherein the α7-nAChR LBD comprises a Q79G amino acid substitution and a P216I amino acid substitution.

9. The modified LGIC of any one of claims 1 to 8, wherein the IPD comprises an amino acid substitution at residue 298.

10. The modified LGIC of claim 9 , wherein the IPD is a GlyR IPD and the amino acid substitution is an A298G substitution.

11. 10. The modified LGIC of claim 9, wherein the IPD is a GABA IPD and the amino acid substitution is a W298A substitution.

12. 2. The modified LGIC of claim 1, wherein the α7-nAChR LBD further comprises an R27D substitution and an E41R substitution, and the modified LGIC subunit selectively binds to another modified LGIC subunit having an R27D substitution and an E41R substitution over binding to an unmodified LGIC subunit.

13. The modified LGIC of any one of claims 1 to 8, 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, and the at least one modified amino acid in the 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 the mouse 5HT3 IPD.

14. 14. The modified LGIC of claim 13, wherein at least one modified amino acid comprises an R425Q substitution, an R429D substitution, and / or an R433A substitution.

15. The modified LGIC of any one of claims 1 to 8, 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, and the at least one modified amino acid in the 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 the human 5HT3 IPD.

16. 16. The modified LGIC of claim 15, wherein at least one modified amino acid comprises an R420Q substitution, an R424D substitution, and / or an R428A substitution.

17. The modified LGIC of any one of claims 1 to 16, wherein the LBD has reduced binding to an endogenous LGIC ligand.

18. 18. The modified LGIC of claim 17, wherein the endogenous LGIC ligand is acetylcholine (ACh).

19. 19. The modified LGIC of claim 18, wherein the modified LGIC has an EC50 for ACh of greater than 20 μM.

20. The modified LGIC of any one of claims 1 to 19, wherein 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, 2-phenyl-7,8,9,10-tetrahydro-6H-6,10-methanoazepino[4,5-g]quinoxaline, 1,4-diazabicyclo[3.2.2]nonane, and varenicline.

21. 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 according to any one of claims 1 to 20; detecting binding between the candidate ligand and the modified LGIC, thereby identifying a ligand that selectively binds to the modified LGIC; A method comprising:

22. 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 LGICs according to any one of claims 1 to 20; 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:

23. A mammalian cell comprising the modified LGIC of any one of claims 1 to 20.

24. A nucleic acid encoding the modified LGIC subunit of any one of claims 1 to 20.

25. 1. A homomeric chimeric ligand-gated ion channel (LGIC) comprising chimeric LGIC subunits, wherein each chimeric LGIC subunit is: an alpha 7 nicotinic acetylcholine receptor ligand binding domain having amino acid substitutions selected from the group consisting of: ii) an L131M amino acid substitution and a Y115F amino acid substitution; iii) a W77F amino acid substitution, a Q79G amino acid substitution, and a G175K amino acid substitution; iv) a Q79G amino acid substitution, a Y115F amino acid substitution, and a G175K amino acid substitution; and v) a Y115F amino acid substitution and a G175K amino acid substitution, in the sequence set forth in residues 23-224 of SEQ ID NO: 1, residues 23-229 of SEQ ID NO: 2, or residues 23-233 of SEQ ID NO: 11, numbered with reference to SEQ ID NO: 1; 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).

26. 1. A homomeric chimeric ligand-gated ion channel (LGIC) comprising chimeric LGIC subunits, wherein each chimeric LGIC subunit is: an alpha 7 nicotinic acetylcholine receptor ligand binding domain having amino acid substitutions selected from the group consisting of: i) an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution; ii) an L131M amino acid substitution and a Y115F amino acid substitution; iii) a W77F amino acid substitution, a Q79G amino acid substitution, and a G175K amino acid substitution; iv) a Q79G amino acid substitution, a Y115F amino acid substitution, and a G175K amino acid substitution; and v) a Y115F amino acid substitution and a G175K amino acid substitution in the sequence set forth in residues 23-224 of SEQ ID NO:1, residues 23-229 of SEQ ID NO:2, or residues 23-233 of SEQ ID NO:11, numbered with reference to SEQ ID NO:1; 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).

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