Modified ligand gated ion channels and methods of use

Abstract

This document relates to materials and methods for controlling ligand gated ion channel (LGIC) activity. For example, a modified LGIC comprising at least one LGIC subunit is provided, the LGIC subunit having a modified ligand binding domain (LBD) and / or a modified ion pore domain (IPD). Also provided are exogenous LGIC ligands that can bind to and activate the modified LGIC, as well as methods of modulating ion transport across a mammalian cell membrane, methods of modulating cellular excitability in a mammalian, and methods of treating a mammalian with channeling disease.

Description

[0001] This application is a divisional application of a Chinese invention patent application filed on July 7, 2017, with application number: 201780054322.6 (PCT application number: PCT / US2017 / 041147), and invention name: “Modified ligand-gated ion channels and methods of use”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Patent Application Serial No. 62 / 359,534, filed on July 7, 2016, and claims the benefit of U.S. Patent Application Serial No. 62 / 486,779, filed on April 18, 2017. The disclosure of the prior application is considered part of (and incorporated by reference into) the disclosure of the present application. Background of the Invention 1. Technical Field

[0005] This document relates to materials and methods for controlling the activity of ligand-gated ion channels (LGICs). For example, this document provides a modified LGIC comprising at least one LGIC subunit having a modified ligand binding domain (LBD) and / or a modified ion pore domain (IPD). An exogenous LGIC ligand is also provided that can bind to and activate the modified LGIC. In some cases, the modified LGIC and the exogenous ligand can be used to treat mammals suffering from channelopathy (e.g., neuropathies or muscle channelopathy). In some cases, the modified LGIC and the exogenous LGIC ligand can be used to regulate (e.g., activate or inhibit) ion transport across mammalian cell membranes. In some cases, the modified LGIC and the exogenous LGIC ligand can be used to regulate (e.g., increase or decrease) the excitability of cells in mammals.

[0006] 2. Background Information

[0007] Ion channels mediate ion flux in cells, which greatly influences their biological functions. A prominent example of this is neurons, where ion channels control electrical signaling between neurons to influence physiology, sensation, behavior, mood, and cognition.

[0008] Different LGICs have different ligand binding properties as well as specific ion conductance properties (Hille 2001 Ion Channels of Excitable Membranes. pp. 814. Sunderland, MA: Sinauer Associates; Kandel et al 2000 Principles of Neural Science. USA: McGraw-Hill Co. 1414 pp). For example, nicotinic acetylcholine receptors (nAChRs) bind the endogenous ligand acetylcholine (ACh), which activates the cation conductance and generally depolarizes the cell, thereby increasing cell excitability. In contrast, glycine receptors (GlyRs) bind the endogenous ligand glycine, which activates the chloride ion conductance and generally reduces the excitability of the cell by hyperpolarization and / or electrical shunting through the cell membrane resistance. Summary of the invention

[0009] The levels of endogenous LGIC agonists such as ACh are not easily controlled.

[0010] This document provides materials and methods for controlling LGIC activity (e.g., increasing the sensitivity of LGIC to exogenous ligands and / or reducing the sensitivity to endogenous ligands such as ACh). For example, this document provides a modified LGIC comprising at least one modified LGIC subunit having an LBD and an IPD and having at least one modified amino acid (e.g., an amino acid substitution). An exogenous LGIC ligand is also provided that can bind to and activate the modified LGIC. In some cases, the modified LGIC and the exogenous ligand can be used to treat mammals suffering from channel diseases (e.g., neural channel diseases or muscle channel diseases). In some cases, the modified LGIC and the exogenous LGIC ligand can be used to regulate (e.g., activate or inhibit) ion transport across mammalian cell membranes. In some cases, the modified LGIC and the exogenous LGIC ligand can be used to regulate (e.g., increase or decrease) the excitability of cells in mammals.

[0011] Having the ability to control LGIC activity provides a unique and unrealized opportunity to achieve control of ion transport in cells. For example, modified LGICs with increased sensitivity to one or more exogenous LGIC ligands can be used to provide temporal and spatial control of ion transport and / or cell excitability based on the delivery of exogenous LGIC ligands. For example, modified LGICs with reduced sensitivity to endogenous LGIC ligands prevent the undesired activation of modified LGICs and allow selective control of modified LGICs by exogenous ligands. In addition, exogenous LGIC ligands with increased efficacy to modified LGICs improve the targeting selectivity of modified LGICs relative to endogenous ion channels. Therefore, the modified LCIG and exogenous LGIC ligands provided herein can be used to achieve therapeutic effects while reducing the side effects of small molecules on unintended targets.

[0012] As described herein, one or more mutations in the modified LGIC can enhance the potency of exogenous LGIC ligands. Mutations of the α7LBD of the α7-GlyR at residue L131 (e.g., substitution of Leu with Gly or Ala) increase the potency of varenicline (16-fold) and tropisetron (3.6-fold) relative to α7-GlyR, while reducing the potency of ACh (-6.4-fold). Mutations of the α7LBD of the α7-GlyR at residues G175 (e.g., G175K) or P216 (e.g., P216I) enhance the potency of Ach, nicotine, tropisetron, varenicline, and other quinoline and tropane agonists. Combining a mutation at residue G175K with a mutation that reduces the potency of the endogenous agonist ACh (e.g., Y115F) yields an α7-GlyR Y115F G175K with increased potency (5.5-fold) for tropisetron and reduced potency (-8-fold) for Ach. In addition, combining mutations at residues 77 (e.g., substitution of Trp with Phe or Tyr) and / or 79 (e.g., substitution of Gln with Gly, Ala, or Ser) and / or 131 (e.g., substitution of Leu with Gly or Ala) and / or 141 (e.g., substitution of Leu with Phe or Pro) in these chimeric channels in the α7 LBD with potency-enhancing mutations at residues G175 (e.g., G175K) or P216 (e.g., P216I) increases the potency of the unique ligand and / or reduces Ach potency. For example, a chimeric α7-GlyRLGIC containing an α7 nAChR LBD (α7 LBD) having a mutation at residue 79 (e.g., substitution of Gln with Gly), a mutation at residue 115 (e.g., substitution of Tyr with Phe), and a mutation at residue 175 (e.g., substitution of Gly with Lys) has greater than 100-fold increased sensitivity to the exogenous tropane LGIC ligand compound 723 (tropane) and decreased Ach sensitivity (-15-fold) relative to the unmodified chimeric α7-GlyR LGIC. Furthermore, the modified LGIC, comprising at least one chimeric LGIC subunit containing an α7 nAChR LBD (α7 LBD) having a mutation at residue 79 (e.g., substitution of Ala, Gly or Ser for Gln) and a GlyR IPD having a mutation at residue 298 (e.g., substitution of Gly for Ala). Additional mutations at residues 27 (e.g., substitution of Asp for Arg) and 41 (e.g., substitution of Arg for Glu) of the α7 LBD reduced binding of the modified chimeric LGIC to unmodified ion channels.Additional mutations at residues 115 (e.g., substitution of Tyr with Phe), 139 (e.g., substitution of Gln with Gly or Leu), 210 (e.g., substitution of Tyr with Phe), 217 (e.g., substitution of Tyr with Phe), and / or 219 (e.g., substitution of Asp with Ala) of the α7 LBD reduce the sensitivity of the chimeric LGIC to the endogenous ligand ACh. These chimeric LGICs allow for highly selective control of cellular function in mammalian cells while minimizing cross-responsiveness with endogenous signaling systems in mammals.

[0013] In general, one aspect of this document features a modified LGIC having at least one modified LGIC subunit comprising an LBD and an IPD having amino acid modifications, wherein an exogenous LGIC ligand activates the modified LGIC. The modified LGIC can be a chimeric LGIC having an LBD from a first LGIC and an IPD from a second LGIC. The LBD can be an α7 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 can be at residue 79 of the α7 LBD, and the amino acid substitution can be Q79A, Q79G, or Q79S. For example, the amino acid substitution at residue 79 of the α7 LBD can be Q79G. The IPD may be a serotonin 3 receptor (5HT3) IPD, a glycine receptor (GlyR) IPD, a γ-aminobutyric acid (GABA) receptor IPD, or an α7-nAChR IPD. The IPD may be a GlyR IPD, and the GlyR IPD may include an amino acid substitution at residue 298 of a chimeric LGIC (e.g., an A298G substitution). The IPD may be a GABA IPD, and the GABA IPD may include an amino acid substitution at residue 298 of a modified LGIC (e.g., a W298A substitution). The modified LGIC may be a chimeric LGIC comprising an α7LBD having a Q79G amino acid substitution and a GlyR IPD having 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-methylene-6H-pyrazino(2,3-h)benzaprozemide and 1,4-diazabicyclo[3.2.2]nonane. When the synthetic exogenous LGIC ligand is tropane, the tropane can be tropisetron, pseudotropisetron, nortropisetron, compound 723, compound 725, compound 737 or compound 745. When the synthesized exogenous LGIC ligand is quinuclidine, 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 synthesized exogenous LGIC ligand is 6,7,8,9-tetrahydro-6,10-methylene-6H-pyrazino(2,3-h)benzapromide, 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 some cases, the LBD can be an α7LBD, and the α7LBD can also include at least one modified amino acid that confers selective binding to another α7LBD having at least one modified amino acid relative to binding to an unmodified LGIC. The unmodified LGIC can be an endogenous LGIC (e.g., an endogenous α7-nAChR). At least one modified amino acid in the α7LBD that confers reduced binding to the unmodified LGIC can 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 can be a 5HT3 IPD, and the 5HT3IPD can include at least one modified amino acid that confers increased ionic conductance to the modified LGIC. The at least one modified amino acid in the 5HT3 IPD that confers increased ionic conductance to the modified LGIC may include an amino acid substitution at amino acid residues at residues 425 (e.g., R425Q substitution), 429 (e.g., R429D substitution), and / or 433 (e.g., R433A substitution).

[0014] In another aspect, the document herein features a modified LGIC having at least one modified LGIC subunit comprising an LBD and an IPD having at least one modified amino acid, wherein at least one modified amino acid in the LBD reduces binding to an endogenous LGIC ligand. The modified LGIC may be a chimeric LGIC having an LBD from a first LGIC and an IPD from a second LGIC. The endogenous LGIC ligand may be ACh. The modified LGIC may have an EC50 of greater than 20 μM for Ach. The at least one modified amino acid may 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 may be a Y115F substitution. When the at least one modified amino acid includes an amino acid substitution at residue 139, the amino acid substitution may be a Q139G or Q139L substitution. When at least one modified amino acid comprises an amino acid substitution at residue 210, the amino acid substitution may be a Y210F substitution. When at least one modified amino acid comprises an amino acid substitution at residue 217, the amino acid substitution may be a Y217F substitution. When at least one modified amino acid comprises an amino acid substitution at residue 219, the amino acid substitution may be a D219A substitution.

[0015] In another aspect, this document features a ligand with increased potency on a modified ligand-gated ion channel (LGIC), wherein the ligand comprises Formula I:

[0016]

[0017] wherein X1, X2 and X3 are each independently CH, CH2, O, NH or NMe; wherein each n can be independently 0 or 1; wherein Y=O or S; wherein A=aromatic substituent; wherein R=H or pyridylmethylene. The aromatic substituent can be 1H-indole, 4-(trifluoromethyl)benzene, 2,5-dimethoxybenzene, 4-chloroaniline, aniline, 5-(trifluoromethyl)pyridin-2-yl, 6-(trifluoromethyl)nicotine, or 4-chlorobenzene.

[0018] In some cases, the LGIC ligand can be quinuclidine and can have the structure shown in Formula II:

[0019]

[0020] wherein X3=O, NH or CH2; wherein Y=O or S; wherein A=aromatic substituent; wherein R=H or pyridylmethylene. The aromatic substituent can 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. Quinucleoside 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.

[0021] In some cases, the LGIC ligand can be tropane, and can have the structure shown in Formula III:

[0022]

[0023] wherein X2=NH or NMe; wherein X3=O, NH or CH2; wherein Y=O or S; wherein A=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.

[0024] In some cases, the LGIC ligand can be 9-azabicyclo[3.3.1]nonane and can have the structure shown in Formula IV:

[0025]

[0026] 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.

[0027] In another aspect, the present document features a ligand having increased potency on a modified ligand-gated ion channel (LGIC), wherein the ligand can be 6,7,8,9-tetrahydro-6,10-methylene-6H-pyrazino(2,3-h)benzaprone and has a structure shown in Formula V:

[0028]

[0029] wherein R can be H or CH3, and wherein A can be H or an aromatic substituent. 6,7,8,9-tetrahydro-6,10-methylene-6H-pyrazino(2,3-h)benzaprozemide can be varenicline, compound 0765 or compound 0770.

[0030] In another aspect, this document features a ligand with increased potency for a modified ligand-gated ion channel (LGIC), wherein the ligand can be 1,4-diazabicyclo[3.2.2]nonane, and can have the structure shown in Formula VI:

[0031]

[0032] Wherein R can be H, F or NO2. 1,4-diazabicyclo[3.2.2]nonane can be 3-(1,4-diazabicyclo[3.2.2]non-4-yl)dibenzo[b,d]thiophene 5,5-dioxide, Compound 0773, or Compound 0774.

[0033] In another aspect, this document features a method of treating a channelopathy in a mammal. The method comprises or consists essentially of administering a modified LGIC to a mammalian 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 comprising at least one modified amino acid; and administering the exogenous ligand to the mammal.The channelopathy can be Bartter syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT), congenital hyperinsulinism, 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 (long QT syndrome (such as Romano-Ward syndrome), short QT syndrome, malignant hyperthermia, mucolipidosis type IV, myasthenia gravis, myotonia congenital, neuromyelitis optica, neuromyotonia, nonsyndromic deafness, paramyotonia congenital, retinitis pigmentosa, Timothy syndrome, tinnitus, seizures, trigeminal neuralgia, and multiple sclerosis.

[0034] In another aspect, this document features a method of 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, the modified LGIC subunit comprising an LBD and an IPD comprising at least one modified amino acid; and administering an exogenous ligand to the mammal. Modulation may include activating or inhibiting ion transport. The cell may be a neuron, a glial cell, a muscle cell, a stem cell, an endocrine cell, or an immune cell. Administration of the modified LGIC to the cell may be in vivo or ex vivo. Administration of the modified LGIC to the cell may include administering a nucleic acid encoding the modified LGIC.

[0035] In another aspect, this document features a method of regulating cell excitability in a mammal. The method comprises or consists essentially of administering a modified LGIC to a mammalian cell, wherein an exogenous LGIC ligand selectively binds to the modified LGIC. The modified LGIC has at least one modified LGIC subunit, the modified LGIC subunit comprising an LBD and an IPD comprising at least one modified amino acid; and administering an exogenous ligand to the mammal. Regulation may include increasing the excitability of the cell or decreasing the excitability of the cell. The cell may be an excitable cell. The cell may be a neuron, a glial cell, a muscle cell, a stem cell, an endocrine cell, or an immune cell. Administration of the modified LGIC to the cell may be in vivo or ex vivo. Administration of the modified LGIC to the cell may include administration of a nucleic acid encoding the modified LGIC.

[0036] In another aspect, the document features a method of regulating cell 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, the modified LGIC subunit comprising an LBD and an IPD comprising at least one modified amino acid; and administering an exogenous ligand to the mammal. Regulation may include increasing cell activity or decreasing cell activity. The activity may be ion transport, passive transport, excitation, inhibition, or exocytosis. The cell may be a neuron, a glial cell, a muscle cell, a stem cell, an endocrine cell, or an immune cell. Administering the modified LGIC to a cell may be in vivo or ex vivo. Administering the modified LGIC to a cell may include administering a nucleic acid encoding the modified LGIC (e.g., via a viral vector, such as an adeno-associated virus, herpes simplex virus, or a lentivirus).

[0037] 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 ligand and the modified LGIC, thereby identifying a ligand that selectively binds to the modified LGIC. The modified LGIC can be a homomeric modified LGIC.

[0038] In another aspect, the document 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 binding between the modified LGIC and the agent that selectively binds to the modified LGIC, thereby detecting the modified LGIC. The agent that selectively binds to the modified LGIC comprises an antibody, a protein (e.g., a bungarus toxin), or a small molecule (e.g., a positron emission tomography (PET) ligand). The agent that selectively binds to the modified LGIC may comprise a detectable marker (e.g., a fluorescent marker, a radioactive marker, or a positron emitting marker).

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Methods and materials for use in the present disclosure are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0040] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1A-1EShown are exemplary amino acid sequences of chimeric LGICs. The mutation of amino acid residue 77 (e.g., W77F or W77Y) results in sensitivity to granisetron and tropisetron. The mutation of amino acid residue 79 (e.g., Q79G) is most effective for several agonists. The mutation of amino acid residue 131 (e.g., L131G, L131A, L131M or L131N) changes the sensitivity to varenicline, tropisetron, granisetron and ACh. When the LBD mutation is combined with the mutation at amino acid residue 298 in GlyR or GABAc IPD, the effect is considerably enhanced. When the α7 nAChR LBD mutation is combined with the mutation at amino acid residues G175 and P216, the effect is also enhanced. Figure 1A ) The amino acid sequence of the α7-5HT3 chimeric receptor (SEQ ID NO: 6), including human α7 nAChR LBD (SEQ ID NO: 1) and mouse 5HT3 IPD (SEQ ID NO: 3) components. Figure 1B ) The amino acid sequence of the α7-GlyR chimeric receptor (SEQ ID NO: 7), including human α7 nAChR LBD (SEQ ID NO: 2) and human GlyR IPD (SEQ ID NO: 5) components. Figure 1C ) The amino acid sequence of the α7-5HT3 chimeric receptor (SEQ ID NO: 8), including human α7 nAChR LBD (SEQ ID NO: 1) and human 5HT3 IPD (SEQ ID NO: 4) components. Figure 1D ) The amino acid sequence of the α7-GABAc chimeric receptor (SEQ ID NO: 10), including human α7 nAChR LBD (SEQ ID NO: 2) and human GABAc IPD (SEQ ID NO: 9) components. Figure 1E ) Amino acid sequence of rat nAChR sequence (SEQ ID NO: 12).

[0043] Figure 2 The effects of tropisetron on α7-5HT3 chimeric LGIC and Figures 1A-1E EC50 of variants of chimeric LGICs with LBD mutations at the indicated positions. Multiple mutations at Gln79 showed similar or improved potency relative to unmodified α7-5HT3 channels (arrows).

[0044] Figure 3A-3B The relative potency of known nAChR agonists on the α7-5HT3 chimeric LGIC is shown. Figure 3A ) Plot of EC50 normalized to unmodified α7-5HT3 chimeric channels (logarithmic scale). *P<0.05, statistically significant change in potency noted (ANOVA followed by Dunn's test). Figure 3B )The chemical structures of nAChR agonists are known.

[0045] Figures 4A-4C The relative potency of known nAChR agonists on α7-GlyR chimeric LGICs is shown. Figure 4A ) EC50 plots of Q79 LBD mutants normalized to the unmodified α7-GlyR chimeric channel (logarithmic scale). Figure 4B ) EC50 plot of the A298G IPD mutation normalized to the unmodified α7-GlyR chimeric channel (logarithmic scale). Figure 4C ) were normalized to the unmodified α7-GlyR chimeric channel and compared with the double mutant channel α7Q79G-GlyR A298G Comparison of α7-GlyR A298G EC50 graphs (logarithmic scale). *P<0.05, statistically significant change in potency noted (ANOVA followed by Dunn's test).

[0046] Figures 5A-5C Schematic structures of LGIC agonists with α7 Q79G -5HT3 and α7 Q79G -GlyR A298G The substitution patterns most compatible with potency enhancement of α7 are shown in Figure 2. A) Generic structures showing properties associated with potency enhancement. B) Specific pharmacophores shown in (A) are quinuclidine, tropane, and 9-azabicyclo[3.3.1]nonane core structures. C) Exemplary synthetic molecules that enhance α7 Q79G -GlyR A298G 、α7 Q79G，Y115F，G175K -GlyR, α7 W77F,Q79G，G175K -GlyR showed high potency.

[0047] Figures 6A-6C Mutations that reduce binding of the chimeric LCIG α7 nAChR LBD to the unmodified LBD are shown. Fig. 6A ) were transfected with two constructs encoding α7-5HT3 with epitope tags (HA and V5) (top) or two constructs encoding α7-5HT3-HA and α7 R21D，E41R Schematic diagram of charge reversal potential conformation of the construct of -5HT3-V5, in which the binding between the two subunits with different epitope tags is unfavorable due to the charge reversal mutation at the subunit interface. Figure 6B ) in expressing α7 with a V5 epitope tag R2ID，E41R Whole-cell recordings in HEK cells expressing -5HT3 revealed a robust response to PNU-282987. Figure 6C) α7-5HT3 LGICs were probed for binding to HA and V5 epitope tags in HEK cells by HA immunoprecipitation (left) or fractionation of total lysates followed by western blotting with anti-HA (top) or anti-V5 antibodies (bottom). In cells co-expressing channels with HA and V5 epitopes, anti-HA IP followed by anti-V5 immunoblotting showed co-immunoprecipitation of each type of unmodified channel, but LBD α7 R2ID，E41R -The charge reversal mutation in 5HT3-V5 was not immunoprecipitated. The MW of α7-5HT3 is approximately 48 kD (arrow).

[0048] Figure 7 showed that chimeric LGICs can be controlled using exogenous ligands. Q79G -GlyR A298G Cortical neurons of chimeric LGIC-transduced mouse brains fired action potentials in response to 40 pA current injection (PRE), which were potently inhibited by 30 nM tropisetron. Neuron firing was restored after washout of tropisetron (WASH).

[0049] Figures 8A-8C The activity of the agonist on a chimeric LGIC with the G175K mutation is shown. Fig. 8A ) Plot of the EC50 of the Q79G G175K LBD mutant against known agonists normalized to the unmodified α7-GlyR chimeric channel (logarithmic scale). Figure 8B ) Plot of EC50 for ACh and Tropisetron for channels with mutations in α7-GlyR chimeric LGIC. Mutations resulting in high potency for Tropisetron and low potency for the endogenous ligand acetylcholine (ACh) are optimal (grey shading). Unmodified: Unmodified α7-GlyR chimeric LGIC. Figure 8C ) from α7 Q79G，Y115F，G175K Action potentials of cortical neurons of mouse brain transduced with -GlyR chimeric LGICs. Neurons fired in response to current injection (PRE) and were potently inhibited by 100 nM tropisetron. Neuronal firing was restored after tropisetron washout (WASH).

[0050] Figures 9A-9D The activity of the agonist on a chimeric LGIC with the L131G mutation is shown. Fig.9A ) Plot of the EC50 of L131 LBD mutants for known agonists normalized to unmodified α7-GlyR chimeric channels (logarithmic scale). Fig. 9B )α7 L131G - Plot of the EC50 of channels with mutations in GlyR chimeric LGICs for ACh and tropisetron. Fig. 9C) The figure shows that the mutations resulting in high potency for varenicline and low potency for the endogenous ligand acetylcholine (ACh) are optimal (grey shading). Unmodified: Unmodified α7-GlyR chimeric LGIC. Fig.9D ) from α7 L131G，Q139L，Y217F Action potentials of cortical neurons of mouse brain transduced with -GlyR chimeric LGICs. Neurons fired in response to current injection (PRE) and were potently inhibited by 10 nM varenicline, even when the injected current was >6-fold greater. Neuronal firing was restored after tropisetron washout (WASH).

[0051] Figures 10A-10B The chemical structures of LGIC agonists are shown. Fig. 10A ) has the same Q79G，Y115F，G175K Chemical structures of LGIC agonists with the most compatible substitution patterns for potency enhancement of -GlyR. Fig. 10B ) has the same L131G，Q139L，Y217F -GlyR or α7 L131G，Q139L，Y217F Chemical structures of LGIC agonists with the most compatible substitution patterns for potency enhancement of -5HT3 HC. DETAILED DESCRIPTION

[0052] This document provides modified LGICs and methods of using the same. For example, this document provides a modified LGIC comprising at least one modified LGIC subunit having an LBD and an IPD and having at least one modified amino acid (e.g., an amino acid substitution). In some cases, the modified LGIC may be a chimeric LGIC. For example, a chimeric LGIC may include an LBD from a first LGIC and an IPD from a second LGIC. In some cases, the modified amino acid may impart pharmacological selectivity to the modified LGIC. For example, the modified amino acid may impart selective binding of an exogenous LGIC ligand to the modified LGIC. For example, the modified amino acid may impart reduced (minimized or eliminated) binding to the modified LGIC relative to an unmodified LGIC subunit (lacking a modified LGIC subunit and / or an endogenous LGIC subunit). For example, the modified amino acid may impart reduced (minimized or eliminated) binding of an endogenous LGIC ligand to the modified LGIC.

[0053] The modified LGICs provided herein can be used, for example, in methods for treating channelopathies (e.g., neural channelopathies or muscle channelopathies). For example, modified LGICs and exogenous LGIC ligands that can bind to and activate modified LGICs can be used to treat mammals suffering from channelopathies. In some cases, modified LGICs and exogenous LGIC ligands can be used to regulate (e.g., activate or inhibit) ion transport across mammalian cell membranes. In some cases, modified LGICs and exogenous LGIC ligands can be used to regulate (e.g., increase or decrease) the excitability of cells in mammals.

[0054] Modified LGIC

[0055] As used herein, a "modified" LGIC is an LGIC comprising at least one LGIC subunit. The modified LGIC subunit may comprise 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 LGICs from any suitable species (e.g., humans, rats, mice, dogs, cats, horses, cattle, goats, pigs, or monkeys). In some cases, the modified LGIC may include at least one chimeric LGIC subunit having a non-naturally occurring combination of an LBD from a first LGIC and an IPD from a second LGIC.

[0056] The modified LGIC can be homomeric (e.g., having any number of identical modified LGIC subunits) or heteromeric (e.g., having at least one modified LGIC subunit and any number of different LGIC subunits). In some cases, the modified LGIC described herein can be a homomeric modified LGIC. The modified LGIC described herein can include any suitable number of modified LGIC subunits. In some cases, the modified LGIC can be a trimer, a tetramer, a pentamer, or a hexamer. For example, the modified LGIC described herein can be a pentamer.

[0057] The modified LGIC subunits described herein can be modifications of any suitable LGIC. LGICs can conduct anions, cations, or both across a cell membrane in response to the binding of a ligand. For example, LGICs can transport sodium (Na+), potassium (K+), calcium (Ca2+), and / or chloride (Cl-) ions across a cell membrane in response to the binding of a ligand. Examples of LGICs include, but are not limited to, Cys loop receptors (e.g., AChRs, such as nAChRs (e.g., muscle nAChRs or neuronal nAChRs), gamma-aminobutyric acid (GABA; e.g., GABA A and GABA A-ρ (also known as GABA C ) receptors, GlyR, GluCl receptors and 5HT3 receptors), ionotropic glutamate receptors (iGluR; such as AMPA receptors, kainate receptors, NMDA receptors and δ receptors), ATP-gated channels (such as P2X) and phosphatidylinositol 4,5-bisphosphate (PIP2)-gated channels. In the case where the modified LGIC described herein is a chimeric LGIC, the chimeric LGIC may include an LBD selected from any appropriate LGIC and an IPD selected from any appropriate LGIC. In the case where the LGIC includes multiple different subunits (e.g., neuronal nAChR includes α4, β2 and α7 subunits), the LBD and / or IPD can be selected from any subunit. For example, the LBD from a nAChR can be an α7 LBD. A representative rat α7 nAChR amino acid sequence (including both the LBD and the IPD) is as follows.

[0058] SEQ ID NO: 12

[0059]

[0060] In some cases, the modified LGIC subunit described herein may include an LBD from an α7 nAChR. Examples of α7 nAChR LBDs include, but are not limited to, a human α7 nAChR LBD having an amino acid sequence as set forth in SEQ ID NO: 1, a human α7 nAChR LBD having an amino acid sequence as set forth in SEQ ID NO: 2, and a human α7 nAChR LBD having an amino acid sequence as set forth in SEQ ID NO: 11. In some 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 some cases, the α7 nAChR LBD can have at least 75% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 11 (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).

[0061] SEQ ID NO: 1

[0062]

[0063] SEQ ID NO: 2

[0064]

[0065] SEQ ID NO: 11

[0066]

[0067] In some cases, the modified LGIC subunit described herein may include an IPD from a 5HT3 receptor. Examples of 5HT3 IPDs include, but are not limited to, a mouse 5HT3 IPD having an amino acid sequence as set forth in SEQ ID NO: 3 and a human 5HT3 IPD having an amino acid sequence as set forth in SEQ ID NO: 4. In some cases, the 5HT3 IPD may be a homolog, an ortholog, or a paralog of the 5HT3 IPD set forth in SEQ ID NO: 3 or SEQ ID NO: 4. In some 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) with SEQ ID NO: 3 or SEQ ID NO: 4.

[0068] SEQ ID NO: 3

[0069]

[0070] SEQ ID NO: 4

[0071]

[0072] In some cases, the modified LGIC subunit described herein may include an IPD from GlyR. Examples of GlyR IPDs include, but are not limited to, a mouse GlyR IPD having an amino acid sequence as shown in SEQ ID NO: 5. In some cases, the GlyRIPD may be a homolog, ortholog, or paralog of the human GlyR IPD shown in SEQ ID NO: 5. In some 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) with SEQ ID NO: 5.

[0073] SEQ ID NO: 5

[0074]

[0075] In some cases, the modified LGIC subunits described herein may include a GABA receptor (e.g., GABA A-ρ , also known as GABA C ) of IPD. GABAA-ρ Examples of IPD include, but are not limited to, human GABA having an amino acid sequence as shown in SEQ ID NO: 9. A-ρ IPD. In some cases, GABA A -ρ IPD may be human GABA shown in SEQ ID NO: 9 A-ρ Homologs, orthologs, or paralogs of IPD. In some cases, GABA A-ρ The IPD may have at least 75% sequence identity to SEQ ID NO:9 (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).

[0076] SEQ ID NO: 9

[0077]

[0078] In calculating the percentage of sequence identity, two sequences are compared and the number of identical matches of amino acid residues between the two sequences is determined. The number of identical matches is divided by the length of the comparison region (i.e., the number of amino acid residues compared) and multiplied by 100 to obtain the percentage sequence identity value. It should be understood that the length of the comparison region can be a portion of one or both sequences, up to the full-length size of the shortest sequence. It should also be understood that a single sequence can be compared with more than one other sequence, and therefore, different percentage sequence identity values ​​can be provided on each comparison region. The comparison of two or more sequences can be performed using the computer program ClustalW and default parameters to determine the percentage of sequence identity, which calculates the best match between the query and one or more subject sequences and aligns them, thereby determining identity, similarity and difference. See, for example, Chenna et al., 2003, Nucleic Acids Res., 31 (13): 3497-500.

[0079] In the case where the modified LGIC subunit described herein is a chimeric LGIC subunit, the chimeric LGIC subunit may include an LBD and an IPD from the same species or an LBD and an IPD from different species. In some cases, the chimeric LGIC subunit may include an LBD from a human LGIC protein and an IPD from a human LGIC protein. For example, a chimeric LGIC subunit may include a human α7 LBD and a human GlyR IPD. In some cases, a chimeric LGIC subunit may include an LBD from a human LGIC protein and an IPD from a mouse LGIC protein. For example, a chimeric LGIC subunit may include a human α7 LBD and a mouse 5HT3 IPD.

[0080] Where the modified LGIC subunits described herein are chimeric LGIC subunits, the chimeric LGIC subunits can include different fusion points connecting the LBD and the 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 Figure 1A and Figure 1C and Figure 1B ).

[0081] The modified LGIC subunits described herein may include an LBD having at least one modified amino acid and / or an IPD having at least one modified amino acid. For example, the modified LGIC subunits described herein may include an α7 LBD having at least 75% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12, and amino acid substitutions at amino acid residues 27, 41, 77, 79, 131, 139, 141, 175, 210, 216, 217, and / or 219. For example, the modified LGIC subunits described herein may include a GlyR IPD having at least 75% sequence identity to the sequence shown 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 subunits described herein may include a GABA. IPD having at least 75% sequence identity to SEQ ID NO: 9, and an α7-GABA C Amino acid substitution at amino acid residue 298 of a chimeric receptor (e.g., SEQ ID NO: 10). In some cases, the modified LGIC subunit described herein may include more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more) amino acid modifications. The modification may be an amino acid substitution. In some 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 to the modified LGIC relative to an unmodified LGIC subunit (lacking a modified LGIC subunit and / or an endogenous LGIC subunit). For example, the modified amino acid may confer reduced (minimized or eliminated) binding of an endogenous LGIC ligand to the modified LGIC.

[0082] In some aspects, the modified LGIC subunit described herein may comprise at least one modified amino acid that confers selective binding to an exogenous LGIC ligand (e.g., enhanced binding or increased potency) to the modified LGIC. Binding to an exogenous LGIC ligand may be selective relative to binding to an endogenous LGIC ligand. A modified LGIC subunit having selective binding to an exogenous LGIC ligand may include any suitable LDB (e.g., α7 LBD). In some aspects, the modified LGIC subunit may include the α7 LBD shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12, and the amino acid modification may be a substitution at amino acid residues 77, 79, 131, 139, 141, 175, and / or 216. In some 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, the modified LGIC subunit described herein can include the α7 LBD shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11 or SEQ ID NO: 12 and having a W77F substitution. In some 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, the modified LGIC subunit described herein can include the α7 LBD having a Q79G substitution. In some 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 some cases, the glycine at amino acid residue 175 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11 or SEQ ID NO: 12 can be substituted with an amino acid residue such as lysine (e.g., G175K), alanine (e.g., G175A), phenylalanine (e.g., G175F), histidine (e.g., G175H), methionine (e.g., G175m), arginine (e.g., G175R), serine (e.g., G175S), valine (e.g., G175V). In some cases, the proline at amino acid residue 216 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11 or SEQ ID NO: 12 can be substituted with an amino acid residue such as isoleucine (e.g., P216I). The modified LGIC subunit having selective binding to an exogenous LGIC ligand can include any suitable IPD (e.g., GlyR IPD or GABA. A -ρ IPD). In some aspects, the modified LGIC subunit may include the GlyR IPD shown in SEQ ID NO: 5, and the amino acid modification may be a substitution at amino acid residue 298 of the α7-GlyR chimeric receptor (e.g., SEQ ID NO: 7). In some cases, the alanine at amino acid residue 298 of SEQ ID NO: 7 may be substituted with an amino acid residue such as glycine (e.g., A298G). In some aspects, the modified LGIC subunit may include the GABA shown in SEQ ID NO: 9 A-ρ IPD, and the amino acid modification can be α7-GABA A-ρ Substitutions at amino acid residue 298 of a chimeric receptor (eg, SEQ ID NO: 10). In some cases, the tryptophan at amino acid residue 298 of SEQ ID NO: 10 can be substituted with an amino acid residue such as alanine (eg, W298A).

[0083] In some cases, the modified LGIC subunits described herein may include more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more) amino acid modifications. For example, the modified LGIC subunits described herein may have at least 75% sequence identity to SEQ ID NO: 7 and may include a Q79G substitution and an A298G substitution. Other examples of modifications that may confer selective binding of exogenous LGIC ligands to the modified LGIC include modifications described elsewhere (see, e.g., US8,435,762).

[0084] A modified LGIC subunit that selectively binds (e.g., has enhanced binding or increased potency) to an exogenous LGIC ligand relative to an endogenous (e.g., canonical) LGIC ligand can also be described as having enhanced potency for the exogenous ligand. In some cases, a modified LGIC subunit that selectively binds to an exogenous LGIC ligand as described herein can have at least a 4-fold (e.g., at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold) enhanced potency for the exogenous ligand. In some cases, the modified LGIC subunits described herein that selectively bind to an exogenous LGIC ligand can have an enhanced potency of about 4-fold to about 200-fold (e.g., about 4-fold to about 200-fold, about 5-fold to about 180-fold, about 6-fold to about 175-fold, about 7-fold to about 150-fold, about 8-fold to about 125-fold, about 9-fold to about 100-fold, about 10-fold to about 90-fold, about 11-fold to about 75-fold, about 12-fold to about 65-fold, about 13-fold to about 50-fold, about 14-fold to about 40-fold, or about 15-fold to about 30-fold) for the exogenous ligand. For example, the modified LGIC subunits described herein that selectively bind to an exogenous LGIC ligand can have an enhanced potency of about 10-fold to about 100-fold for the exogenous ligand. For example, the modified LGIC subunits described herein that selectively bind to an exogenous LGIC ligand can have an enhanced potency of about 10-fold to about 20-fold for the exogenous ligand.

[0085] In some aspects, the modified LGIC subunit described herein may include at least one modified amino acid that imparts reduced (e.g., minimized or eliminated) binding to the modified LGIC relative to the unmodified LGIC subunit. The binding to the modified LGIC subunit with the same modification may be selective relative to the binding to the unmodified LGIC subunit. The unmodified LGIC subunit may be a LGIC subunit lacking a modification that imparts reduced binding to the modified LGIC relative to the unmodified LGIC subunit, or the unmodified LGIC may be an endogenous LGIC subunit. The modification that imparts reduced binding to the modified LGIC relative to the unmodified LGIC subunit may be a charge reversal modification. The modified LGIC subunit with reduced binding relative to the unmodified LGIC subunit may include any suitable LBD (e.g., α7 LBD). In some aspects, the modified LGIC subunit can include the α7 LBD shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12, and the amino acid modification can be a substitution at amino acid residue 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 can 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 some cases, the modified LGIC subunit described herein can include an α7 LBD having an R27D substitution and E41R.

[0086] In some aspects, the modified LGIC subunit described herein may include 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 endogenous LGIC ligand binding may include any suitable IPD (e.g., GlyR LBD). For example, the modified LGIC subunit may include the α7 LBD shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11, or SEQ ID NO: 12, and the amino acid modification may be a substitution at amino acid residues 115, 131, 139, 210, 217, and / or 219. In some 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 some 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 some 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 some 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 some 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 some 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).

[0087] In some aspects, the modified LGIC subunit described herein may include at least one modified amino acid that imparts increased ionic conductance to the modified LGIC. In some cases, the modified LGIC subunit may include the 5HT3 IPD shown in SEQ ID NO: 3, and the amino acid modification may be a substitution at amino acid residues 425, 429, and / or 433. For example, the modified LGIC subunit described herein may include a 5HT3 IPD having an R425Q substitution, an R429D substitution, and an R433A substitution. In some cases, the modified LGIC subunit may include the 5HT3IPD shown in SEQ ID NO: 4, and the amino acid modification may be a substitution at amino acid residues 420, 424, and / or 428. For example, the modified LGIC subunit described herein may include a 5HT3 IPD having an R420Q substitution, an R424D substitution, and an R428A substitution.

[0088] In some cases, a modified LGIC described herein can include at least one chimeric α7-5HT3 LGIC subunit (SEQ ID NO:6) having a human α7 nAChR LBD (SEQ ID NO:1) having a Q79G amino acid substitution and a Y115F amino acid substitution, and a murine 5HT3 IPD (SEQ ID NO:3).

[0089] In some cases, a modified LGIC described herein can include at least one chimeric α7-5HT3 LGIC subunit (SEQ ID NO:6) having a human α7 nAChR LBD (SEQ ID NO:1) having a Q79G amino acid substitution and a Q139G amino acid substitution, and a murine 5HT3 IPD (SEQ ID NO:3).

[0090] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 1) having a Q79G amino acid substitution and a Y115F amino acid substitution, and a human GlyR IPD (SEQ ID NO: 5) having an A298G amino acid substitution.

[0091] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) having a Q79G amino acid substitution and a Q139G amino acid substitution, and a human GlyR IPD (SEQ ID NO: 5) having an A298G amino acid substitution.

[0092] In some cases, the modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID No: 2) having an R27D amino acid substitution, an E41R amino acid substitution, a Q79G amino acid substitution, and a Y115F amino acid substitution, and a human GlyR IPD (SEQ ID NO: 5) having an A298G amino acid substitution.

[0093] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) containing a substitution at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N), and a human GlyR IPD (SEQ ID NO: 5).

[0094] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7nAChR LBD (SEQ ID NO: 2) containing substitutions at amino acid residues 131 (e.g., L131G, L131A, L131M, or L131N) and Y115 (e.g., Y115F), and a human G1yR IPD (SEQ ID NO: 5).

[0095] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) containing 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).

[0096] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) containing 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).

[0097] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) containing 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).

[0098] In some cases, a modified LGIC described herein can include at least one chimeric α7-5HT3 LGIC subunit having a human α7 nAChR LBD (SEQ ID NO: 2) containing a substitution at amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N), and a human 5HT3 IPD (SEQ ID NO: 4).

[0099] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) containing a substitution at amino acid residue 175 (e.g., G175K), and a human GlyR IPD (SEQ ID NO: 5).

[0100] In some cases, a modified LGIC described herein can include at least one chimeric α7-5HT3 LGIC subunit having a human α7 nAChR LBD (SEQ ID NO: 2) containing substitutions at amino acid residues 131 (e.g., L131G, L131A, L131M, or L131N) and 139 (e.g., Q139L), and a human 5HT3 IPD (SEQ ID NO: 4) having a R420Q substitution, a R424D substitution, and a R428A substitution.

[0101] In some cases, a modified LGIC described herein can include at least one chimeric α7-5HT3 LGIC subunit having a human α7nAChR LBD (SEQ ID NO: 2) containing substitutions at amino acid residues 131 (e.g., L131G, L131A, L131M, or L131N) and 139 (e.g., Q139L) and 217 (e.g., Y217F), and a human 5HT3 IPD (SEQ ID NO: 4) having a R420Q substitution, a R424D substitution, and a R428A substitution.

[0102] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) containing substitutions at amino acid residues 175 (e.g., G175K) and 115 (e.g., Y115F), and a human GlyR IPD (SEQ ID NO: 5).

[0103] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) containing 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).

[0104] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) containing 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).

[0105] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) containing a substitution at amino acid residue 216 (e.g., P216I), and a human GlyR IPD (SEQ ID NO: 5).

[0106] In some cases, a modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 7) having a human α7 nAChR LBD (SEQ ID NO: 2) containing substitutions at amino acid residues 216 (e.g., P216I) and 79 (e.g., Q79G), and a human GlyR IPD (SEQ ID NO: 5).

[0107] In some cases, the modified LGIC described herein can include at least one chimeric α7-GlyR LGIC subunit (SEQ ID NO: 10) having a human α7 nAChR LBD (SEQ ID NO: 2) containing a substitution at amino acid residue 131 (e.g., L131A, L131G, L131M, L131N, L131Q, L131V, or L131F), and a human GABA c IPD (SEQ ID NO: 9).

[0108] In the case where the LBD and / or IPD is a homologue, orthologue or paralogue of a sequence described herein (e.g., SEQ ID NOs: 1-5 and / or 9), it is understood that reference to a particular modified amino acid residue can be transferred to the corresponding amino acid in the homologue, orthologue or paralogue. For example, residues 425, 429 and 433 in the murine 5HT3 IPD set forth in SEQ ID NO: 3 correspond to residues 420, 424 and 428 in the human 5HT3 IPD set forth in SEQ ID NO: 4, and the R425Q, R429D and R433A substitutions in the murine 5HT3 IPD correspond to the R420Q, R424D and R428A substitutions in the human 5HT3 IPD.

[0109] Any method can be used to obtain the modified LGIC subunits described herein. In some cases, peptide synthesis methods can be used to prepare the modified LGIC subunits described herein. Examples of peptide synthesis methods include, but are not limited to, liquid phase peptide synthesis and solid phase peptide synthesis. In some cases, protein biosynthesis methods can be used to prepare 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 phosphorylation mimetic peptides provided herein. Similar modified LGIC subunits (e.g., modified subunits having substantially the same modifications and / or having substantially the same amino acid sequences) will self-assemble through interactions between LBDs to form modified LGICs.

[0110] This document also provides nucleic acids encoding the modified LGIC subunits described herein and 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)). 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 regulated promoter (e.g., inducible, tissue-specific, cell type-specific (e.g., neuron-specific, muscle-specific, glial-specific) and neural subtype-specific). Examples of promoters that can be used to drive expression of nucleic acids encoding the modified LGIC subunits described herein include, but are not limited to, synaptophysin, CAMKII, CMV, CAG, enolase, TRPV1, POMC, NPY, AGRP, MCH, and Orexin promoters. In some cases, nucleic acids encoding the modified LGIC subunits described herein can be operably linked to neuron-specific promoters.

[0111] This document also provides cells (e.g., mammalian cells) having a modified LGIC as described herein. Mammalian cells having a modified LGIC as described herein can be obtained by any suitable method. In some cases, a preassembled modified LGIC can be provided to the cell. In some cases, a nucleic acid encoding a modified LGIC subunit as described herein can be provided to the cell under conditions where the modified LGIC subunit is translated and where multiple (e.g., 3, 4, 5, 6 or more) modified LGIC subunits can be assembled into a modified LGIC as described herein.

[0112] LGIC ligand

[0113] This document also provides LGIC ligands that can bind to and activate the modified LGIC described herein. The LGIC ligands that can bind to and activate the modified LGIC described herein can be exogenous or endogenous. The LGIC ligands that can bind to and activate the modified LGIC described herein can be naturally occurring or synthetic. The LGIC ligands that can bind to and activate the modified LGIC described herein can be canonical or non-canonical. The LGIC ligands that can bind to and activate the modified LGIC described herein can be agonists or antagonists. In some cases, the LGIC ligand is an exogenous LGIC agonist. Examples of LGIC ligands include, but are not limited to, ACh, nicotine, epibatatine, cytisine, RS56812, tropisetron, nortropisetron, PNU-282987, PHA-543613, Compound 0353, Compound 0354, Compound 0436, Compound 0676, Compound 702, Compound 723, Compound 725, granisetron, dihydrovermectin, mequitazine, promazine, varenicline, Compound 765, Compound 770, 3-(1,4-diazabicyclo[3.2.2]non-4-yl)dibenzo[b,d]thiophene 5,5-dioxide, Compound 773, and Compound 774 (see, e.g., Figure 3B , Figure 5C , Fig. 10A and Fig. 10B ).

[0114] LGIC ligands that can bind to and activate a modified LGIC described herein can have selective binding (e.g., enhanced binding or increased potency) for a modified LGIC described herein. In some cases, LGIC ligands that can bind to and activate a modified LGIC described herein do not bind to and activate an endogenous receptor. LGIC ligands that selectively bind to and activate a modified LGIC described herein (e.g., a modified LGIC having at least one amino acid modification that confers pharmacological selectivity for the modified LGIC) relative to an unmodified LGIC ligand can also be described as having enhanced potency for the modified LGIC. In some cases, the modified LGIC subunit described herein that selectively binds an exogenous LGIC ligand can have 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) enhanced potency over the modified LGIC. For example, an LGIC ligand that selectively binds to and activates a modified LGIC can have an enhanced potency for the modified LGIC by about 10-fold to about 300-fold (e.g., about 10-fold to about 250-fold, about 10-fold to about 200-fold, about 10-fold to about 150-fold, about 10-fold to about 100-fold, about 25-fold to about 300-fold, about 50-fold to about 300-fold, about 100-fold to about 300-fold, about 200-fold to about 300-fold, about 25-fold to about 250-fold, about 50-fold to about 200-fold, or about 100-fold to about 150-fold). In some cases, a LGIC ligand that binds to and activates a modified LGIC described herein can have a ligand potency of less than 25 nM (e.g., less than 22 nM, less than 20 nM, less than 17 nM, less than 15 nM, less than 13 nM, less than 12 nM, less than 11 nM, less than 10 nM, less than 5 nM, less than 2 nM, or less than 1 nM). For example, a LGIC ligand that binds to and activates a modified LGIC described herein can have a ligand potency of less than 15 nM. In some cases, a LGIC ligand can have an EC50 of less than 25 nM for a modified LGIC subunit described herein (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 example, a LGIC ligand (e.g., tropisetron) can have an EC50 of less than 25 nM for a modified LGIC subunit described herein (e.g., α7 Q79G -GlyR A298G) can have an EC50 of about 11 nM. For example, a LGIC ligand (e.g., nortropisetron) can have an EC50 of about 11 nM for a modified LGIC subunit described herein (e.g., α7 Q79GY115F -GlyR A298G ) can have an EC50 of about 13 nM. In some cases, the LGIC ligand can have an EC50 of greater than 20 μM (e.g., greater than 22 μM, greater than 25 μM, greater than 35 μM, greater than 50, greater than 65 μM, greater than 80 μM, or greater than 100 μM) for a modified LGIC subunit described herein. For example, a LGIC ligand (e.g., ACh) can have an EC50 of about 13 nM for a modified LGIC subunit described herein (e.g., α7 Q79G，Y115F -GlyR A298G ) can have an EC50 greater than 100 μM.

[0115] In some aspects, the LGIC ligand can be a synthetic ligand capable of binding to and activating the modified LGIC described herein, which 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.

[0116] LGIC ligands that can target and activate the modified LGIC described herein can have Formula I:

[0117]

[0118] Wherein X1 and X2 can be independently CH, CH2, O, NH or NMe; each n can be independently 0 or 1; Y can be O or S; A can be an aromatic substituent; and R can be H or pyridylmethylene. 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.

[0119] The LGIC ligand that can bind to and activate the modified LGIC described herein can be quinuclidine. Quinuclidine can have the structure of Formula II:

[0120]

[0121] 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 pyridylmethylene. 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 quinoline include, but are not limited to, compounds PNU-282987, PHA-543613, 0456, 0434, 0436, 0354, 0353, 0295, 0296, and 0676 (see, e.g., Figure 5C , Table 3 and Table 6).

[0122] The LGIC ligand that can bind to and activate the modified LGIC described herein can be tropane. Tropane can have the structure of Formula III:

[0123]

[0124] Wherein X2 can be NH or NMe; X3 can be O, NH or CH2; Y can be O or S; and 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, pseudo-tropisetron, nortropisetron, compound 737, and compound 745 (see, e.g., Figure 5C , Table 3 and Table 6).

[0125] The LGIC ligand that can bind to and activate the modified LGIC described herein can be 9-azabicyclo[3.3.1]nonane. 9-azabicyclo[3.3.1]nonane can have a structure of Formula IV:

[0126]

[0127] 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. An example of an aromatic substituent is, but is not limited to, 4-chloro-benzene. Examples of 9-azabicyclo[3.3.1]nonane include, but are not limited to, Compound 0536, Compound 0749, Compound 0751, Compound 0760, and Compound 0763 (see, e.g., Figure 5C , Table 3 and Table 6).

[0128] In some cases, the LGIC ligand can be 6,7,8,9-tetrahydro-6,10-methylene-6H-pyrazino(2,3-h)benzapine and can have the structure shown in Formula V:

[0129]

[0130] wherein R = H or CH3; and wherein A = H or an aromatic substituent. Examples of 6,7,8,9-tetrahydro-6,10-methylene-6H-pyrazino(2,3-h)benzazepines include, but are not limited to, varenicline, compound 0765, and compound 0770 (see, e.g. Fig. 10A , Table 3 and Table 9).

[0131] In some cases, the LGIC ligand can be 1,4-diazabicyclo[3.2.2]nonane and can have the structure shown in Formula VI:

[0132]

[0133] Wherein R = H, F, NO2. Examples of 1,4-diazabicyclo[3.2.2]nonane include, but are not limited to, 3-(1,4-diazabicyclo[3.2.2]non-4-yl)dibenzo[b,d]thiophene 5,5-dioxide, Compound 0773 and Compound 0774 (see, e.g. Fig. 10B , Table 6 and Table 9).

[0134] How to use

[0135] This document also provides methods of using the modified LGICs and LGIC ligands described herein, which can bind to and activate the modified LGICs as described herein.LGIC ligands that can bind to and activate the modified LGICs can be used to activate the modified LGICs with temporal and / or spatial control based on the delivery of the ligand.

[0136] In some aspects, the modified LGICs described herein and LGIC ligands that can bind to and activate the modified LGICs as described herein can be used to identify ligands that selectively bind to the modified LGICs described herein. For example, such screening methods can include providing one or more candidate ligands to the modified LGICs described herein, and detecting binding between the candidate ligands and the modified LGICs.

[0137] Any suitable method can be used to detect the binding between the candidate ligand and the modified LGIC, and any suitable method can be used to detect the activity of the modified LGIC. For example, the ability of the ligand to bind and activate the modified LGIC can be measured by an assay including, but not limited to, a membrane potential (MP) assay (e.g., a fluorescent MP assay), a radioactive binding assay, and / or voltage clamp measurements of peak current and sustained current.

[0138] In some aspects, the modified LGICs described herein and LGIC ligands that can bind to and activate the modified LGICs as described herein can be used to treat mammals with channelopathies (e.g., neural channelopathies or muscle channelopathies). For example, a mammal with a channelopathic disease can be treated by administering a modified LGIC described herein, followed by administering a LGIC ligand that can bind to and activate the modified LGIC. For example, a mammal with a channelopathic disease 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 nAChRLBD (SEQ ID NO: 2) containing an R27D amino acid substitution, an E41R amino acid substitution, a Q79G amino acid substitution, and a Y115F amino acid substitution, and a human GlyR IPD (SEQ ID NO: 5) having an A298G amino acid substitution), followed by administering tropisetron. For example, a mammal having a channelopathy can be treated by administering a modified LGIC described herein, comprising 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) having 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.

[0139] 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 as 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 as described herein. In some cases, dogs, cats, horses, cattle, 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 as described herein.

[0140] Any suitable method may be used to identify mammals having a channelopathy and / or mammals at risk of developing a channelopathy. For example, genetic testing may be used to identify mammals having a channelopathy and / or mammals at risk of developing a channelopathy.

[0141] Once a mammal has been identified as having a channelopathy and / or at risk of developing a channelopathy, the mammal can be administered or instructed to self-administer a modified LGIC as described herein, and then administered or instructed to self-administer a LGIC ligand that can bind to and activate a modified LGIC as described herein. The modified LGIC as described herein and the LGIC ligand that can bind to and activate a modified LGIC as described herein can be administered together or can be administered separately.

[0142] When treating a mammal suffering from a channelopathy and / or a mammal at risk of developing a channelopathy using materials and methods as described herein, the channelopathy can be any channelopathy. As used herein, a channelopathy can be any disease or condition caused by abnormal ion channel function and / or abnormal ligand function, or any disease or condition that can be alleviated by regulating ion channel function and / or altered cell ion flux (e.g., calcium ion flux). Channelopathy can be congenital or acquired. Examples of channelopathies include, but are not limited to, Bartter syndrome, Brugada syndrome, catecholamine-dependent ventricular tachycardia (CPVT), congenital hyperinsulinemia, cystic fibrosis, Dravet syndrome, paroxysmal ataxia, erythromelalgia, generalized epilepsy (e.g., with febrile seizures), familial hemiplegic migraine, fibromyalgia, hyperkalemic periodic paralysis, hypokalemic periodic paralysis, Lambert 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 deafness, paramyotonia congenita, retinitis pigmentosa, timothy syndrome, tinnitus, seizures, trigeminal neuralgia, and multiple sclerosis. Alternatively, or additionally, the materials and methods described herein may be used in other applications including, but not limited to, pain treatment, cancer cell therapy, appetite control, spasticity treatment, dystonia treatment, tremor treatment, and movement disorder treatment.

[0143] In some cases, the modified LGICs described herein and the LGIC ligands that can bind to and activate the modified LGICs as described herein can be used to regulate the activity of cells. The cell activity regulated using the modified LGICs described herein and the LGIC ligands that can bind to and activate the modified LGICs as described herein can be any cell activity. Examples of cell activities include, but are not limited to, active transport (e.g., ion transport), passive transport, excitation, inhibition, ion flux (e.g., calcium ion flux), and exocytosis. Cell activity can be increased or decreased. For example, the modified LGICs described herein and the LGIC ligands that can bind to and activate the modified LGICs as described herein can be used to regulate (e.g., increase) ion transport across the cell membrane. For example, the modified LGICs described herein and the LGIC ligands that can bind to and activate the modified LGICs as described herein can be used to regulate (e.g., increase) the excitability of cells.

[0144] 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., a neutrophil, an eosinophil, a basophil, a lymphocyte, and a monocyte), an endocrine cell, or a stem cell (e.g., an embryonic stem cell). In some cases, the cell can be an excitable cell. The cell can be in vivo or in vitro.

[0145] The modified LGIC described herein can be administered by any suitable method. The modified LGIC can be administered as a modified LGIC subunit or as a preassembled modified LGIC. The modified LGIC can be administered as a nucleic acid encoding the modified LGIC. The modified LGIC can be administered as a nucleic acid encoding the modified LGIC subunit described herein. For example, the nucleic acid can be delivered as a naked nucleic acid or 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 a dividing cell or a non-dividing cell. 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 viral vectors), cosmids, and artificial chromosomes (e.g., yeast artificial chromosomes or bacterial artificial chromosomes). In some cases, the nucleic acid encoding the modified LGIC subunit described herein can be expressed by an adeno-associated viral vector.

[0146] The modified LGIC described herein can be detected by any appropriate method (e.g., to confirm its presence in a cell). In some cases, a reagent that selectively binds to the modified LGIC can be used to detect the modified LGIC. Examples of reagents that can be used to bind to the modified LGIC described herein include, but are not limited to, antibodies, proteins (e.g., bungarus toxins), and small molecule ligands (e.g., PET ligands). Reagents that selectively bind to the modified LGIC can include detectable markers (e.g., fluorescent markers, radioactive markers, positron emission markers, and enzyme markers). Methods for detecting LGIC expression in cells can include fluorescence imaging, autoradiography, functional MRI, PET, and SPECT.

[0147] The modified LGICs described herein and LGIC ligands that can bind to and activate a modified LGIC as 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 for treating the channelopathy. For example, a combination therapy for treating a mammal having a channelopathy as described herein can include administering a modified LGIC as described herein and a LGIC ligand that can bind to and activate a modified LGIC as described herein, and treating with acetazolaminde, dichlorphenamide, mexilitine, glucose, calcium gluconate, L-DOPA, muscle stimulation, spinal stimulation, brain stimulation, and / or nerve stimulation.

[0148] In embodiments where the modified LGIC described herein and the LGIC ligands that can bind and activate the modified LGIC as described herein are combined with other agents / therapies for treating channel diseases, one or more additional agents can be administered simultaneously or independently. For example, the modified LGIC described herein and the LGIC ligands that can bind and activate the modified LGIC as described herein are first administered, and then one or more additional agents are administered, or vice versa. In embodiments where the modified LGIC described herein and the LGIC ligands that can bind and activate the modified LGIC as described herein are used in combination with one or more additional therapies for treating channel diseases, one or more additional therapies can be administered simultaneously or independently while administering the modified LGIC described herein and the LGIC ligands that can bind and activate the modified LGIC as described herein. For example, the modified LGIC described herein and the LGIC ligands that can bind and activate the modified LGIC as described herein can be administered before, during, or after performing one or more additional therapies.

[0149] In some cases, the modified LGICs described herein and / or LGIC ligands that can bind to and activate the modified LGICs as described herein can be formulated into pharmaceutically acceptable compositions for administration to mammals suffering from channel diseases or at risk of developing channel diseases. For example, a therapeutically effective amount of a modified LGIC as described herein (e.g., a nucleic acid encoding a modified LGIC as described herein) and / or a LGIC ligand that can bind to and activate the modified LGICs as described herein can be formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents. Pharmaceutical compositions can be formulated for administration in solid or liquid form, including but not limited to sterile solutions, suspensions, sustained release formulations, tablets, capsules, pills, powders, and granules.

[0150] 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 phosphates, 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 silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin.

[0151] Pharmaceutical compositions containing modified LGICs as described herein and / or LGIC ligands that can bind and activate modified LGICs as 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 modified LGICs as described herein (e.g., nucleic acids encoding modified LGICs as described herein) and / or LGIC ligands that can bind and activate modified LGICs as 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 can contain antioxidants, buffers, bacteriostats, and solutes that make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents. Compositions for inhalation can be delivered using, for example, an inhaler, a nebulizer, and / or a dry powder inhaler. The formulations can be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets.

[0152] A pharmaceutically acceptable composition comprising a therapeutically effective amount of a modified LGIC as described herein (e.g., a nucleic acid encoding a modified LGIC as described herein) and / or a LGIC ligand that can bind to and activate a modified LGIC as described herein can be administered topically or systemically. In some cases, a composition comprising a therapeutically effective amount of a modified LGIC as described herein (e.g., a nucleic acid encoding a modified LGIC as described herein) and / or a LGIC ligand that can bind to and activate a modified LGIC as described herein can be administered systemically by intravenous or oral administration to a mammal (e.g., a human) or by inhalation by a mammal (e.g., a human). In some cases, a composition comprising a therapeutically effective amount of a modified LGIC as described herein (e.g., a nucleic acid encoding a modified LGIC as described herein) and / or a LGIC ligand that can bind to and activate a modified LGIC as described herein can be administered topically to a target tissue of a mammal (e.g., a human) by transdermal, subcutaneous, intramuscular, intracranial, or open surgical administration (e.g., injection).

[0153] The effective dosage 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 treatments (eg, use of other agents), and the judgment of the treating physician.

[0154] The frequency of administration can be any frequency that improves the symptoms of channel diseases without producing significant toxicity to mammals. For example, the frequency of administration can be from about once a week to about three times a day, from about twice a month to about six times a day, or from about twice a week to about once a day. The frequency of administration can remain constant or can change during the treatment duration. The process of treating with a composition containing a therapeutically effective amount of a modified LGIC as described herein (e.g., a nucleic acid encoding a modified LGIC as described herein) and / or a composition that can bind and activate a LGIC ligand of a modified LGIC as described herein can include a rest period. For example, a composition containing a therapeutically effective amount of a modified LGIC as described herein (e.g., a nucleic acid encoding a modified LGIC as described herein) and / or a composition that can bind and activate a LGIC ligand of a modified LGIC as described herein can be administered daily over a two-week period, 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 frequency of administration for a particular application. For example, the effective amount, duration of treatment, use of multiple therapeutic agents, route of administration, and severity of channel diseases can require an increase or decrease in the frequency of administration.

[0155] The effective duration of administration of a composition containing a therapeutically effective amount of a modified LGIC as described herein (e.g., a nucleic acid encoding a modified LGIC as described herein) and / or a LGIC ligand that can bind to and activate a modified LGIC as described herein can be any duration that can improve channelopathy symptoms without causing significant toxicity to a mammal. For example, the effective duration can vary from a few days to a few weeks, a few months, or a few years. In some cases, the effective duration for treating a channelopathy can range from a duration of about one month to about 10 years. Various factors can affect the actual effective duration for a particular treatment. For example, the effective duration can vary with the frequency of administration, the effective amount, the use of a variety of therapeutic agents, the route of administration, and the severity of the channelopathy being treated.

[0156] In some cases, the course of treatment and the mammal being treated for a channelopathy may be monitored for symptoms.Symptoms of a channelopathy may be monitored using any suitable method.

[0157] The present invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0158] Example

[0159] Example 1: Potency-enhancing ligand binding domain mutations

[0160] A panel of 41 α7-5HT3 chimeric channels with mutant LBDs were screened against a panel of 51 clinically used drugs that are chemically similar to nicotinic receptor agonists. Figures 1A-1E The screening revealed that Gln in the α7 nAChR LBD 79 , which enhances the potency of the known nAChR agonist tropisetron ( Figure 2 These mutations (Q79A, Q79G, Q79S) reduce the size of the amino acid side chains. Some mutant ion channel-ligand combinations gave up to 12-fold improvement in potency (Table 1, Figure 3A ). The canonical α7 nAChR agonists, Ach, nicotine, epibatidine, and the anti-smoking drug varenicline were not significantly affected by the Q79A, Q79G, or Q79S mutations. However, a subset of α7 nAChR agonists showed enhanced potency in the presence of some mutations. Cytisine, RS56812, tropisetron, nortropisetron, and PNU-282987 showed significantly improved potency against α7Q79G-5HT3. In addition, nortropisetron and PNU-282987 showed significant potency against α7Q79G-5HT3, respectively. Q79A -5HT3 and α7 Q79S-5HT3 significantly enhanced potency. In general, agonists based on quinine or tropane pharmacophores with linked aromatic structures that interact with the complementary binding surface of the ligand binding domain show improved potency when Gln79 is substituted with smaller amino acid residues Ala, Gly or Ser. For most agonists, α7 Q79G -5HT3 is the most preferred mutant chimeric ion channel.

[0161] Table 1. Potency of nAChR agonists on chimeric cation channels mutated at Gln79 in HEK cells. Mean EC50 with SEM in brackets (μM).

[0162]

[0163]

[0164] Use of these mutant LBDs generated α7-G1yR chimeric channels that had up to 6-fold enhanced potency for most of these ligands ( Figure 4A ). As with mutations in α7-5HT3, these mutations at Gln79 did not significantly affect the potency of ACh, nicotine, epibatidine, varenicline, or cytisine. However, tropisetron, nortropisetron, and RS56812 showed significant effects on α7 Q79G Similar to the LBD mutation of α7-5HT3, nortropisetron has a significant effect on α7 Q79A -GlyR has significantly enhanced potency, and PNU-282987 shows Q79S -GlyR. For most agonists, α7 Q79G -GlyR is the most preferred mutant chimeric ion channel.

[0165] Another relationship observed in the small molecule screen is that mutations at Trp77 W77F -5HT3 (EC50: 1.2 μM), α7 W77Y -5HT3 (EC50: 1.1 μM) and α7 W77F -GlyR (EC50: 0.66 μM) receptors conferring agonist activity to the drug granisetron. Granisetron is a 5HT3 receptor antagonist that does not activate α7-5HT3 or α7-G1yR.

[0166] These results show that mutation of Q79 (to A, G, or S) in the α7 nAChR LBD enhances the binding of known LGIC ligands to the modified LGIC.

[0167] Example 2: Potency-enhancing ion pore domain mutations

[0168] The enhanced potency of the allosteric agonist dihydrotestosterone was tested on α7-GlyR channels with IPD mutations previously established in full-length glycine receptor channels (T258S and A288G, GlyR numbering; equivalent to T268S and A298G in α68-GlyR numbering). T268S The channels have a substantial ligand-free open probability, which makes them unsuitable for ligand-controlled cellular manipulation. A298G Mutations in , which effectively enhance the potency of dihydrochloride at the full-length glycine receptor, result in modest changes in the probability of opening in the absence of ligand; therefore, activity against a panel of known agonists was examined for this channel. For the canonical agonists ACh, nicotine, and epibatidine, as well as for varenicline and tropisetron, agonist potency was not significantly different at α7-GlyR. A298G A subset of α7 nAChR agonists did show up to a modest 4-fold increase in potency: RS56812, cytisine, PNU-282987, and nortropisetron were significantly more potent. Thus, the effect of the IPDA298G mutation improves ligand potency but is ligand structure dependent and less effective than mutations in the LBD.

[0169] The Q79G mutation in the LBD and the A298G IPD mutation in α7-GlyR were examined (Table 2). Q79G -GlyR A298G The results were synergistic, with up to 18-fold potency enhancement for α7 nAChR agonists relative to α7-GlyR. The enhancement for 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 such as ACh, nicotine, epibatidine, varenicline, and cytisine were found to be highly potent at both α7-GlyR and α7. Q79G -GlyR A298G Thus, the combination of the LBD mutation Q79G and the IPD mutation A298G produces a synergistic effect, with the potency of some, but not all, nicotinic agonists greatly increased by about 10-20 fold.

[0170] Table 2. Potency of nAChR agonists on mutant chimeric chloride channels. Mean EC50 and SEM in parentheses (μM) of agonist activity in HEK cells expressing chimeric channels.

[0171]

[0172] These results show 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 enhances the selective binding of known LGIC ligands to the modified LGIC.

[0173] Example 3: Molecules exhibiting enhanced efficacy

[0174] Based on α7 Q79G -GlyR A298G Based on the structure activity relationship of known agonists that showed enhanced potency in the absence of quinuclidine, tropane, or 9-azabicyclo[3.3.1]nonane pharmacophores with one or more aromatic side chain substituents, various synthetic molecules were tested. In addition, the known α7 nAChR agonist PHA-543613 (Walker et al. 2006, Wishka et al. 2006) was tested and showed enhanced potency for α7. Q79G -GlyR A298G These molecules typically showed enhanced potency 10- to 100-fold (Table 3), indicating that for these pharmacophores, a range of structural features are associated with targeting α7 Q79G -GlyR A298G The improved effectiveness is compatible.

[0175] These results show that the modified LGIC can be activated by quinuclidine-containing and tropane-containing synthetic LGIC ligands.

[0176]

[0177] Example 4: Mutations that reduce acetylcholine responsiveness

[0178] Compared to other nAChR isoforms, α7 nAChRs have relatively low sensitivity to ACh, and potency-enhancing mutations for tropane and quinuclidine ligands do not substantially alter the potency of acetylcholine at these channels. Therefore, chimeric channels were further modified to reduce the acetylcholine responsiveness of these channels. In some cases, acetylcholine responsiveness was substantially reduced to over 100 μM with the additional LBD mutations Y115F and Q139G, which only modestly reduced the potency of certain agonists to α7. Q79G，Y115F -5HT3, α7 Q79G，Q139G -5HT3, α7 Q79G，Q139G -GlyR A298 、α7 Q79G，Y115F -GlyR A298G For example, α7 Q79G，Y115F -GlyR A298GThe EC50 for nortropisetron was 13 nM and the EC50 for ACh was >100 μM (Table 4).

[0179] Table 4. Potency of nAChR agonists on mutant chimeric chloride channels with low acetylcholine responsiveness.

[0180] Mean EC50 and SEM in brackets (μM) for activity in HEK cells expressing the chimeric channels.

[0181]

[0182] These results show that the Y115F and / or Q139G mutations in the α7 nAChR LBD reduce the binding of the endogenous LGIC ligand Ach to the modified LGIC.

[0183] Example 5: Mutations that reduce binding to endogenous receptor subunits

[0184] 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 binding to endogenous α7 nAChR subunits and / or unwanted binding of chimeric channels, potential intersubunit salt bridges were identified by examining the crystal structure of the acetylcholine binding protein and identifying nearby intersubunit residues with opposite charges that also had cognate ionic amino acids in the α7 nAChR receptor LBD. Charge reversal mutations (converting the acidic member of the 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 the charge reversal mutations ( Fig. 6A ). Chimeric LGIC subunits with charge reversal mutations are able to selectively assemble with each other without interacting with unmodified channels, such as endogenous α7 nAChR. Double mutations of R27D, E41R in the α7 nAChR LBD produce functional channels ( Figure 6B ). Co-expression of these charge-reversal channels with α7-5HT3 channels with unmodified sequences showed that the charge-reversal subunits did not co-immunoprecipitate with the unmodified channels ( Figure 6C ). Combined with potency enhancing mutations and acetylcholine blocking mutations to give chimeric channel α7 R27D,E41R，Q79G，Y115F -GlyR A298G It was revealed that some agonists retained high potency against their cognate agonists (Table 4, right column).

[0185] These results show that the R27D and E41R mutations in the α7 nAChR LBD reduce the binding of modified LGIC subunits to other modified and / or endogenous LGIC subunits.

[0186] Example 6: LBD mutations that increase ligand potency

[0187] Testing the Gly of α7 nAChR LBD in α7-GlyR chimeric channels 175 and Pro 216 Mutation of Gly 175 Mutation to Lys(α7 G175K -GlyR) showed an increase in the potency of ACh (5-fold) (Table 5). G175K -GlyR, it was also found that nicotine potency was enhanced 10-fold relative to the unmodified α7-G1yR chimeric channel (Table 5). 216 Mutation to Ile(α7 P216I -GlyR) did not substantially change ACh potency (Table 5). However, relative to unmodified α7-GlyR, α7 P216I _GlyR showed increased nicotine potency >4-fold (Table 5). α7 G175K -GlyR and α7 P216I These potency-enhancing mutations in -GlyR also affect the potency of several other α7-GlyR agonists by up to 30-fold (Table 5). G175K -G1yR, a greater than 10-fold potency enhancement relative to the α7-GlyR was observed for the clinically used drugs tropisetron, varenicline, cytisine, granisetron, and epibatidine. P216I _G1yR, the potency was enhanced by about 3 times (Table 5).

[0188]

[0189] For use in ACh-producing organisms, it is important to reduce the endogenous ACh potency at these channels constituted by the α7 nAChR LBD. The mutation G175K can be further combined with other mutations that reduce sensitivity to ACh, such as Y115F and Y210F. Y115F，G175K -GlyR, high potency of agonists based on tropane or quinuclidine core structures was found for tropisetron, granisetron, nortropisetron, PNU-282987, and PHA-543613, and greatly reduced potency for varenicline and cytisine (Table 5). G175K，Y210F -GlyR, the potency of most agonists was considerably reduced, but an enhanced potency was observed for granisetron (Table 5).

[0190] To develop channels with reduced ACh responsiveness but high potency to other agonists, α7 G175K -GlyR in combination with other mutations that increase the potency of specific agonists. Combination with W77F reduces ACh potency, and α7W77F，G175K -GlyR showed increased potency for granisetron, nortropisetron, and tropisetron relative to α7-GlyR, but not for PNU282-987, varenicline, cytisine, or PHA-543613 (Table 5). The combination of G175K and Q79G reduced ACh potency, and α7 Q79G，G175K -GlyR showed increased potency for nortropisetron, PHA-543613, and tropisetron (Table 5). However, no such potency enhancement was observed for other agonists, such as PNU282-987 or varenicline. α7 G175K ,Q139L -GlyR decreased the potency of ACh and increased the potency of nortropisetron and tropisetron (Table 5).

[0191] Further reductions in ACh potency were achieved while maintaining the high potency of synthetic agonists, including those based on tropane and quinuclidine core structures, by incorporating mutations at W77F, Q79G, L141F, Y115F, G175K, and Y210F in various combinations. Q79G，Y115F，G175K -GlyR reduced ACh responsiveness while maintaining a robust response to tropisetron (Table 5). Y115F,G175K These mutations also enhanced responsiveness to other tropane and quinuclidine core structures, particularly quinuclidine thioureas 702 and 703 and tropane esters 723, 725, 726, 736, 737, 738, and 745, relative to α7-5HT3 (representing endogenous α7 nAChR activity) (Table 6). Q79G，Y115F，G175K -GlyR also showed high sensitivity to dihydroavidin (Table 5). α7 W77F,Q79G，G175K -GlyR decreased ACh responsiveness while maintaining high potency responses to tropisetron and nortropisetron (Table 5). α7 W77F,Q79G，G175K -GlyR also showed enhanced potency against additional tropane-based core structures (e.g., compounds 723 and 725) as well as the clinically used drugs mequitazine and promazine (Table 6). W77F，G175K，Y210F -GlyR reduced ACh responsiveness but significantly improved the potency of granisetron (Table 5). α7 L141F，Y115F，G175K -GlyR reduces ACh responsiveness and confers sensitivity to granisetron (Table 5). α7 Q79G，Q139L，G175K -GlyR had reduced ACh responsiveness but showed a potent response to nortropisetron (Table 5).

[0192]

[0193]

[0194]

[0195] α7 a175K -G1yR and α7 P216I The mutations at α79G, Y115F, and G175K are also compatible with the non-binding mutations R27D, E41R, and the GlyR IPD mutation A298G, which further enhance the ligand potency of granisetron, epibatidine, varenicline, cytisine, PNU-282987, tropisetron, nortropisetron, and PHA-543613 (Table 7). R27D，E41R，Q79G，Y115F，G175K The potency of 702, 723, 725 and 726 was further improved, with low ACh responsiveness (Table 6).

[0196] Table 7. Provides the G175K and A298G mutations at the α7G1yR chimeric channel and v7GABAc (also known as GABA A -ρ) channels, the agonist potency of W298A was enhanced.

[0197]

[0198] nd = not determined; brackets: SEM

[0199] In #7 Y115F Gly-GlyR chimeric channels 175 Additional amino acid substitutions at α7 also enhanced agonist potency. Y115F The potency of the -GlyR chimeric channel for tropisetron was also 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).

[0200] Table 8. Agonist potency enhancement by G175 mutation at the α7GlyRY115F chimeric channel.

[0201]

[0202] nd = not determined; brackets: SEM

[0203] DISCOVERLeu 131 Mutations to smaller amino acids reduce the potency of the canonical agonists ACh and nicotine, while significantly increasing the potency of varenicline, tropisetron, and several other agonists. α7 L131A -GlyR and α7 L131G-GlyR has reduced ACh responsiveness (6-fold) and enhanced potency for varenicline (8-fold and 17-fold, respectively) and tropisetron (2.5-fold and 3.6-fold, respectively) (Table 9). α7 L131G -5HT3 HCs have reduced ACh responsiveness (5-fold) and enhanced potency to varenicline (16-fold) and tropisetron (2.3-fold) ( Fig.9A and Table 9). α7 L131G，Q139L -GlyR and α7 L131G，Y217F -GlyR showed similar potency enhancement for varenicline relative to α7-GlyR (21-fold), but also reduced ACh sensitivity (-11-fold and -13-fold, respectively). Q79S,L131G -GlyR further improved the potency for varenicline (89-fold) and tropisetron (15-fold). L131G，Q139L，Y217F -GlyR showed the greatest improvement in potency for varenicline (387-fold) and also showed decreased potency for ACh (13-fold) ( Fig. 9B and Table 9). α7 L131G，Q139L，Y217F -GlyR also showed extremely high potency for compound 770 (0.001 μM), compound 773 (0.00034 μM) and compound 774 (0.00013 μM). Figures 10A-10B ). Relative to α7-GlyR, α7 Q79S，L131G，Q139L -GlyR also improved the potency for varenicline (31-fold) and tropisetron (3-fold), but decreased the potency for ACh (9-fold) ( Fig. 9B and Table 9). α7 L131M -GlyR, α7 L131Q -GlyR and α7 L131V -GlyR reduced the potency of ACh, but enhanced the potency of tropisetron, nortropisetron, PHA-543613, and granisetron (Table 9). L131F -GlyR substantially reduced the efficacy of ACh but did not improve the efficacy of other agonists (Table 8). α7 L131G -GABA C Substantially reduced ACh potency but did not improve the potency of other agonists (Table 9). α7 L131G，Q139L，Y217F -5HT3 HC (Table 9) improved the potency of varenicline by 131-fold relative to a7-5HT3 (Table 1). L131G，Q139L，Y217F -5HT3 HC also showed high potency against compound 770 (0.007 μM), compound 773 (0.002 μM) and compound 774 (0.004 μM) (Table 8).

[0204]

[0205]

[0206] Example 7: Chimeric LGICs in Neurons

[0207] The code α7 Q79G _G1yR A298G AAV or DNA plasmids containing nucleic acid of α7Q79GY115F, G175K-GlyR chimeric LGIC were transduced into mouse cortical neurons. Low concentrations of tropisetron (30 nM or 100 nM) were applied to mouse cortical neurons. Neuronal activity was silenced by applying low concentrations of agonists ( Figure 7 and Figure 8C ).

[0208] A DNA plasmid containing nucleic acid encoding the α7L131G, Q139L, Y217F-GlyR chimeric LGIC was transfected into mouse cortical neurons. A low concentration of varenicline (10 nM) was applied to mouse cortical neurons. Neuronal activity was silenced by applying a low concentration of an agonist ( Fig. 9C ).

[0209] These results show that modified LGIC activity can be controlled in neurons using low concentrations of the LGIC ligands tropisetron and varenicline.

[0210] Example 8: Chimeric LGICs in Therapy

[0211] Chemical genetics (chemogenetic) tools provide an attractive strategy for drug and gene combination therapy. This is because it is possible to use exogenously delivered ion channels selectively engaged by administering drugs, using the same ion channels and ligands, to regulate cell function in a consistent manner across different cell types in a variety of indications. The identification of ion channels gated by well-tolerated clinically used drugs is particularly attractive for potentially extending chemical genetics to human therapeutic uses.

[0212] For the drug tropisetron, we found that it activated α7 with an EC50 of 11 nM. Q79G -G1yR A298G , which is similar to the reported IC50 of 10 nM tropisetron for its therapeutic target 5HT3 receptor (Combrink et al 2009 Pharmacological reports: PR 61: 785-97).

[0213] Other Implementations

[0214] It should be understood that although the disclosure has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

[0215] The present invention provides the following contents.

[0216] 1. A modified ligand-gated ion channel (LGIC), comprising at least one modified LGIC subunit, wherein the modified LGIC subunit comprises:

[0217] comprising a ligand binding domain (LBD) with amino acid modifications, and

[0218] Ion pore domain (IPD).

[0219] 2. The modified LGIC of claim 1, wherein the modified LGIC is a chimeric LGIC comprising an LBD from a first LGIC and an IPD from a second LGIC.

[0220] 3. The modified LGIC of claim 1, wherein the LBD is an α7 nicotinic acetylcholine receptor (α7-nAChR) LBD.

[0221] 4. The modified LGIC of claim 3, wherein the amino acid modification comprises an amino acid substitution at one or more amino acid residues selected from the group consisting of residues 77, 79, 115, 131, 139, 141, 175, 210, 216, 217 and 219 of the α7-nAChR LBD.

[0222] 5. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 77 of the α7-nAChR LBD, and wherein the amino acid substitution is selected from the group consisting of W77F and W77Y.

[0223] 6. The modified LGIC of item 4, wherein the amino acid substitution is at residue 79 of the α7-nAChR LBD, and wherein the amino acid substitution is selected from the group consisting of Q79A, Q79G and Q79S.

[0224] 7. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 115 of the α7-nAChR LBD, and wherein the amino acid substitution is a Y115F substitution.

[0225] 8. The modified LGIC of item 4, wherein the amino acid substitution is at residue 131 of the α7-nAChR LBD, and wherein the amino acid substitution is selected from the group consisting of L131A, L131G, L131M and L131N.

[0226] 9. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 139 of the α7-nAChR LBD, and wherein the amino acid substitution is selected from the group consisting of Q139G and Q139L.

[0227] 10. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 175 of the α7-nAChR LBD, and wherein the amino acid substitution is selected from the group consisting of G175A, G175F, G175H, G175K, G175M, G175R, G175S and G175V.

[0228] 11. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 210 of the α7-nAChR LBD, and wherein the amino acid substitution is a Y210F substitution.

[0229] 12. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 216 of the α7-nAChR LBD, and wherein the amino acid substitution is a P216I substitution.

[0230] 13. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 217 of the α7-nAChR LBD, and wherein the amino acid substitution is a Y217F substitution.

[0231] 14. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 219 of the α7-nAChR LBD, and wherein the amino acid substitution is a D219A substitution.

[0232] 15. The modified LGIC of claim 4, wherein the α7-nAChR LBD comprises an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution.

[0233] 16. The modified LGIC of claim 4, wherein the α7-nAChR LBD comprises an L131M amino acid substitution and a Y115F amino acid substitution.

[0234] 17. The modified LGIC of claim 4, wherein the α7-nAChR LBD comprises a W77F amino acid substitution, a Q79G amino acid substitution, and a G175K amino acid substitution.

[0235] 18. The modified LGIC of claim 4, wherein the α7-nAChR LBD comprises a Q79G amino acid substitution, a Y115F amino acid substitution, and a G175K amino acid substitution.

[0236] 19. The modified LGIC of claim 4, wherein the α7-nAChR LBD comprises a Y115F amino acid substitution and a G175K amino acid substitution.

[0237] 20. The modified LGIC of claim 4, wherein the α7-nAChR LBD comprises a Q79G amino acid substitution and a 216I amino acid substitution.

[0238] 21. The modified LGIC of claim 1, 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 γ-aminobutyric acid (GABA) receptor IPD, and an α7 nicotinic acetylcholine receptor (α7-nAChR) IPD.

[0239] 22. The modified LGIC of claim 21, wherein the IPD comprises an amino acid substitution at residue 298.

[0240] 23. The modified LGIC of claim 22, wherein the IPD is a GlyR IPD, and wherein the amino acid substitution is an A298G substitution.

[0241] 24. The modified LGIC of claim 22, wherein the IPD is a GABA IPD, and wherein the amino acid substitution is a W298A substitution.

[0242] 25. The modified LGIC of claim 1, wherein an exogenous LGIC ligand activates the modified LGIC, and 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-methylene-6H-pyrazino(2,3-h)benzaprozemide and 1,4-diazabicyclo[3.2.2]nonane.

[0243] 26. The modified LGIC of claim 25, wherein the synthetic exogenous LGIC ligand is tropane, and wherein the tropane is selected from the group consisting of tropisetron, pseudotropisetron, nortropisetron, Compound 723, Compound 725, Compound 737, and Compound 745.

[0244] 27. The modified LGIC of claim 25, wherein the synthetic exogenous LGIC ligand is quinuclidine, wherein the quinuclidine is selected from the group consisting of PNU-282987, PHA-543613, compound 0456, compound 0434, compound 0436, compound 0354, compound 0353, compound 0295, compound 0296, compound 0536, compound 0676, and compound 702.

[0245] 28. The modified LGIC of claim 25, wherein the synthesized exogenous LGIC ligand is 9-azabicyclo[3.3.1]nonane, and wherein the 9-azabicyclo[3.3.1]nonane is compound 536.

[0246] 29. The modified LGIC of claim 25, wherein the synthetic exogenous LGIC ligand is 6,7,8,9-tetrahydro-6,10-methylene-6H-pyrazino(2,3-h)benzodiazepine, and wherein the 6,7,8,9-tetrahydro-6,10-methylene-6H-pyrazino(2,3-h)benzodiazepine is selected from the group consisting of varenicline, compound 765 and compound 770.

[0247] 30. The modified LGIC of claim 25, wherein the synthesized exogenous LGIC ligand is 1,4-diazabicyclo[3.2.2]nonane, and 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 773, and Compound 774.

[0248] 31. A modified LGIC according to item 1, wherein the LBD is an α7-nAChR LBD, and wherein the α7-nAChR LBD further comprises at least one modified amino acid that confers selective binding to another α7-nAChR LBD having the at least one modified amino acid relative to binding to an unmodified LGIC.

[0249] 32. The modified LGIC of claim 31, wherein the unmodified LGIC is an endogenous LGIC.

[0250] 33. The modified LGIC of claim 32, wherein the endogenous LGIC is an endogenous α7-nAChR.

[0251] 34. The modified LGIC of claim 31, wherein the at least one modified amino acid that confers selective binding comprises an amino acid substitution at an amino acid residue at residue 27 and / or residue 41 of the α7-nAChR LBD.

[0252] 35. The modified LGIC of claim 34, wherein at least one of the modified amino acids comprises an R27D substitution and / or an E41R substitution.

[0253] 36. The modified LGIC of claim 1, wherein the IPD is a rat 5HT3 IPD, and wherein the rat 5HT3 IPD further comprises at least one modified amino acid that confers increased ionic conductance to the modified LGIC.

[0254] 37. The modified LGIC of claim 36, wherein at least one modified amino acid in the mouse 5HT3 IPD that confers increased ionic conductance to the modified LGIC comprises an amino acid substitution at amino acid residues at residues 425, 429 and / or 433 of the mouse 5HT3 IPD.

[0255] 38. The modified LGIC of claim 37, wherein at least one of the modified amino acids comprises a R425Q substitution, a R429D substitution and / or a R433A substitution.

[0256] 39. The modified LGIC of claim 1, wherein the IPD is a human 5HT3 IPD, and wherein the human 5HT3 IPD further comprises at least one modified amino acid that confers increased ionic conductance to the modified LGIC.

[0257] 40. The modified LGIC of claim 39, wherein at least one modified amino acid in the human 5HT3 IPD that confers increased ionic conductance to the modified LGIC comprises an amino acid substitution at amino acid residues at residues 420, 424 and / or 428 of the human 5HT3 IPD.

[0258] 41. The modified LGIC of claim 40, wherein at least one of the modified amino acids comprises a R420Q substitution, a R424D substitution and / or a R428A substitution.

[0259] 42. The modified LGIC of claim 1, wherein the LBD has reduced binding to endogenous LGIC ligands.

[0260] 43. The modified LGIC of claim 42, wherein the endogenous LGIC ligand is acetylcholine (ACh).

[0261] 44. The modified LGIC of claim 43, wherein the modified LGIC has an EC50 for Ach greater than 20 μM.

[0262] 45. A ligand having increased potency on a modified ligand-gated ion channel (LGIC), wherein the ligand comprises Formula I:

[0263]

[0264] wherein X1, X2 and X3 are each independently CH, CH2, O, NH or NMe;

[0265] Wherein each n is independently 0 or 1;

[0266] Where Y = O or S;

[0267] wherein A = an aromatic substituent; and

[0268] Wherein R=H or pyridylmethylene.

[0269] 46. ​​The ligand of claim 45, wherein the aromatic substituent is selected from the group consisting of 1H-indole, 4-(trifluoromethyl)benzene, 2,5-dimethoxybenzene, 4-chloroaniline, aniline, 5-(trifluoromethyl)pyridin-2-yl, 6-(trifluoromethyl)nicotine and 4-chloro-benzene.

[0270] 47. The ligand of claim 45, wherein the ligand is a quinoline having formula II:

[0271]

[0272] Wherein X3=O, NH or CH2;

[0273] Where Y = O or S;

[0274] wherein A = an aromatic substituent; and

[0275] Wherein R=H or pyridylmethylene.

[0276] 48. The ligand of claim 47, wherein the aromatic substituent is selected from the group consisting of 1H-indole, 4-(trifluoromethyl)benzene, 4-chlorobenzene, 2,5-dimethoxybenzene, 4-(trifluoromethyl)benzene, 4-chloroaniline, aniline, 5-(trifluoromethyl)pyridin-2-yl, 6-(trifluoromethyl)nicotine, 3-chloro-4-fluorobenzene and 1H-indole.

[0277] 49. The ligand of item 47, wherein the quinuclidine is selected from the group consisting of PNU-282987, PHA-543613, compound 0456, compound 0434, compound 0436, compound 0354, compound 0353, compound 0295, compound 0296, compound 0536, compound 0676 and compound 702.

[0278] 50. The ligand of claim 45, wherein the ligand is a tropane having formula III:

[0279]

[0280] Where X2 = NH or NMe;

[0281] Wherein X3=O, NH or CH2;

[0282] Where Y = O or S; and

[0283] Where A = aromatic substituent.

[0284] 51. The ligand of item 50, wherein the 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.

[0285] 52. The ligand of claim 50, wherein the tropane is selected from the group consisting of tropisetron, pseudotropisetron, nortropisetron, Compound 723, Compound 725, Compound 737, and Compound 745.

[0286] 53. The ligand of claim 45, wherein the ligand is 9-azabicyclo[3.3.1]nonane having formula IV:

[0287]

[0288] Where X2 = NH or NMe;

[0289] Wherein X3=O, NH or CH;

[0290] Where Y = O or S; and

[0291] Where A = aromatic substituent.

[0292] 54. The ligand of item 54, wherein the aromatic substituent is selected from the group consisting of 4-chloro-benzene, 1H-indole, 1H-indazole, 7-methoxy-1H-indazole.

[0293] 55. The ligand of item 54, wherein the 9-azabicyclo[3.3.1]nonane is selected from the following group: Compound 0536, Compound 0749, Compound 0751, Compound 0760 and Compound 0763.

[0294] 56. A ligand having increased potency on a modified ligand-gated ion channel (LGIC), wherein the ligand comprises Formula V:

[0295]

[0296] Wherein R=H or CH3; and

[0297] Wherein A=H or an aromatic substituent.

[0298] 57. The ligand of item 56, wherein 6,7,8,9-tetrahydro-6,10-methylene-6H-pyrazino(2,3-h)benzaprozene is selected from the following group: varenicline, compound 0765 and compound 0770.

[0299] 58. A ligand having increased potency on a modified ligand-gated ion channel (LGIC), wherein the ligand comprises Formula VI:

[0300]

[0301] Wherein R=H, F or NO2.

[0302] 59. The ligand of item 58, wherein 1,4-diazabicyclo[3.2.2]nonane is selected from the following group: 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-dioxide, Compound 0773 and Compound 0774.

[0303] 60. A method of treating a channelopathy in a mammal, the method comprising:

[0304] 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 comprising:

[0305] a ligand binding domain comprising at least one modified amino acid, and

[0306] an ion pore domain; and

[0307] The exogenous ligand is administered to the mammal.

[0308] 61. The method of claim 60, wherein the channelopathy is selected from the group consisting of Bartter syndrome, Brugada syndrome, catecholamine-dependent ventricular tachycardia (CPVT), congenital hyperinsulinemia, cystic fibrosis, Dravet syndrome, paroxysmal ataxia, erythromelalgia, generalized epilepsy (e.g., with febrile seizures), familial hemiplegic migraine, fibromyalgia, hyperkalemic periodic paralysis, hypokalemic periodic paralysis, Lambert 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 deafness, paramyotonia congenita, retinitis pigmentosa, timothy syndrome, tinnitus, seizures, trigeminal neuralgia, and multiple sclerosis.

[0309] 62. A method for regulating ion transport across a mammalian cell membrane, the method comprising:

[0310] administering to the cell a modified ligand-gated ion channel (LGIC), wherein an exogenous LGIC ligand selectively binds to the modified LGIC, the modified LGIC comprising at least one modified LGIC subunit comprising:

[0311] a ligand binding domain comprising at least one modified amino acid, and

[0312] an ion pore domain; and

[0313] The exogenous ligand is administered to the mammal.

[0314] 63. The method of claim 62, wherein said regulation comprises activating ion transport.

[0315] 64. The method of claim 62, wherein said regulation comprises inhibiting ion transport.

[0316] 65. The method of claim 62, wherein the cells are selected from the group consisting of neurons, glial cells, muscle cells, stem cells, endocrine cells, and immune cells.

[0317] 66. The method of claim 62, wherein administering the modified LGIC to the cell comprises administering it in vivo.

[0318] 67. The method of claim 62, wherein administering the modified LGIC to the cell comprises ex vivo administration.

[0319] 68. A method of regulating cellular excitability in a mammal, the method comprising:

[0320] administering a modified ligand-gated ion channel (LGIC) to cells from the mammal, wherein an exogenous LGIC ligand selectively binds to the modified LGIC, the modified LGIC comprising at least one modified LGIC subunit comprising:

[0321] a ligand binding domain comprising at least one modified amino acid, and

[0322] an ion pore domain; and

[0323] The exogenous ligand is administered to the mammal.

[0324] 69. The method of claim 68, wherein the regulation comprises increasing the excitability of the cells.

[0325] 70. The method of claim 68, wherein the regulation comprises reducing the excitability of the cells.

[0326] 71. The method of claim 68, wherein the cell is an excitable cell.

[0327] 72. The method of claim 68, wherein the cells are selected from the group consisting of neurons, glial cells, muscle cells, stem cells, endocrine cells, and immune cells.

[0328] 73. The method of claim 68, wherein administering the modified LGIC to the cell comprises in vivo administration.

[0329] 74. The method of claim 68, wherein administering the modified LGIC to the cell comprises ex vivo administration.

[0330] 75. A method of modulating cellular activity in a mammal, the method comprising:

[0331] administering to the cell a modified ligand-gated ion channel (LGIC), wherein an exogenous LGIC ligand selectively binds to the modified LGIC, the modified LGIC comprising at least one modified LGIC subunit comprising:

[0332] a ligand binding domain comprising at least one modified amino acid, and

[0333] an ion pore domain; and

[0334] The exogenous ligand is administered to the mammal.

[0335] 76. The method of claim 75, wherein the regulation comprises increasing the activity of the cells.

[0336] 77. The method of claim 75, wherein the regulation comprises reducing the activity of the cells.

[0337] 78. The method of claim 75, wherein the activity is selected from the group consisting of ion transport, passive transport, excitation, inhibition, and exocytosis.

[0338] 79. The method of claim 75, wherein the cells are selected from the group consisting of neurons, glial cells, muscle cells, stem cells, endocrine cells, and immune cells.

[0339] 80. The method of claim 75, wherein administering the modified LGIC to cells from the mammal comprises administering it in vivo.

[0340] 81. The method of claim 75, wherein administering the modified LGIC to cells from the mammal comprises ex vivo administration.

[0341] 82. The modified LGIC of any one of items 60, 62, 68 or 75, wherein the modified LGIC is a chimeric LGIC comprising an LBD from a first LGIC and an IPD from a second LGIC.

[0342] 83. The method of any one of item 82, wherein the chimeric LGIC is a homomeric chimeric LGIC.

[0343] 84. The method of any one of items 60, 62, 68 or 75, wherein administering the modified LGIC to the cell comprises administering a nucleic acid encoding the modified LGIC.

[0344] 85. The method of claim 84, wherein the modified LGIC comprises a sequence that is at least 85% identical to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:10.

[0345] 86. The method of claim 85, wherein the modified LGIC comprises a sequence that is at least 90% identical to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 10.

[0346] 87. The method of claim 86, wherein the modified LGIC comprises a sequence that is at least 95% identical to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 10.

[0347] 88. The method of item 87, wherein the modified LGIC comprises the sequence shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:10.

[0348] 89. The method of any one of items 60, 62, 68 or 75, wherein the LBD is an α7 nicotinic acetylcholine receptor (α7-nAChR) LBD.

[0349] 90. The method of claim 89, wherein at least one modified amino acid in the oα7-nAChR LBD comprises at least one amino acid substitution at an 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.

[0350] 91. The method of claim 90, wherein the amino acid substitution is at residue 77 of the α7-nAChR LBD, and wherein the amino acid substitution is selected from the group consisting of W77F and W77Y.

[0351] 92. The method of claim 90, wherein the amino acid substitution is at residue 79 of the α7-nAChR LBD, and wherein the amino acid substitution is selected from the group consisting of Q79A, Q79G, and Q79S.

[0352] 93. The method of claim 90, wherein the amino acid substitution is at residue 115 of the α7-nAChR LBD, and wherein the amino acid substitution is a Y115F substitution.

[0353] 94. The method of claim 90, wherein the amino acid substitution is at residue 131 of the α7-nAChR LBD, and wherein the amino acid substitution is selected from the group consisting of L131A, L131G, L131M, and L131N.

[0354] 95. The method of claim 90, wherein the amino acid substitution is at residue 139 of the α7-nAChR LBD, and wherein the amino acid substitution is a Q139G or Q139L substitution.

[0355] 96. The method of claim 90, wherein the amino acid substitution is at residue 175 of the α7-nAChR LBD, and wherein the amino acid substitution is selected from the group consisting of G175A, G175F, G175H, G175K, G175M, G175R, G175S, and G175V.

[0356] 97. The method of claim 90, wherein the amino acid substitution is at residue 210 of the α7-nAChR LBD, and wherein the amino acid substitution is a Y210F substitution.

[0357] 98. The method of claim 90, wherein the amino acid substitution is at residue 216 of the α7-nAChR LBD, and wherein the amino acid substitution is P216I.

[0358] 99. The method of claim 90, wherein the amino acid substitution is at residue 217 of the α7-nAChR LBD, and wherein the amino acid substitution is Y217F.

[0359] 100. The method of claim 90, wherein the amino acid substitution is at residue 219 of the α7-nAChR LBD, and wherein the amino acid substitution is D219A.

[0360] 101. The method of any one of items 60, 62, 68 or 75, wherein the IPD comprises at least one modified amino acid.

[0361] 102. The method of item 101, wherein the IPD is selected from the group consisting of a serotonin 3 receptor (5HT3) IPD, a glycine receptor (GlyR) IPD, a GABA receptor IPD, and an α7 nicotinic acetylcholine receptor (α7-nAChR) IPD.

[0362] 103. The method of claim 102, wherein the IPD is a GlyR IPD, and wherein the at least one modified amino acid comprises an amino acid substitution at residue 298 of the modified LGIC.

[0363] 104. The method of claim 103, wherein the amino acid substitution at residue 298 of the modified LGIC is an A298G substitution.

[0364] 105. The modified LGIC of claim 102, wherein the IPD is a GABA IPD, and wherein the at least one modified amino acid in the GABA IPD comprises an amino acid substitution at residue 298 of the modified LGIC.

[0365] 106. The modified LGIC of claim 105, wherein the amino acid substitution at residue 298 of the chimeric LGIC is a W298A substitution.

[0366] 107. The modified LGIC of any one of items 60, 62, 68 or 75, wherein the exogenous LGIC ligand is a synthetic exogenous LGIC ligand.

[0367] 108. The method of claim 107, wherein the synthetic exogenous LGIC ligand is selected from the group consisting of tropane, quinuclidine, 9-azabicyclo[3.3.1]nonane, 1,4-diazabicyclo[3.2.2]nonane and 6,7,8,9-tetrahydro-6,10-methylene-6H-pyrazinyl(2,3-h)phenylpropenazole.

[0368] 109. The method of claim 108, wherein the synthetic exogenous LGIC ligand is tropane, and wherein the tropane is selected from the group consisting of tropanesetron, pseudotropanesetron, nortropanesetron, Compound 723, Compound 725, Compound 737, and Compound 745.

[0369] 110. The method of claim 108, wherein the synthesized exogenous LGIC ligand is quinuclidine, and wherein the quinuclidine is selected from the group consisting of PNU-282987, PHA-543613, compound 0456, compound 0434, compound 0436, compound 0354, compound 0353, compound 0295, compound 0296, compound 0536, compound 0676, and compound 702.

[0370] 111. The method of claim 108, wherein the synthesized exogenous LGIC ligand is 9-azabicyclo[3.3.1]nonane, and wherein the 9-azabicyclo[3.3.1]nonane is selected from the group consisting of Compound 0536, Compound 0749, Compound 0751, Compound 0760, and Compound 0763.

[0371] 112. The method of claim 108, wherein the synthesized exogenous LGIC ligand is 1,4-diazabicyclo[3.2.2]nonane, and 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.

[0372] 113. The method of claim 108, wherein the synthetic exogenous LGIC ligand is 6,7,8,9-tetrahydro-6,10-methylene-6H-pyrazino(2,3-h)benzodiazepine, and wherein 6,7,8,9-tetrahydro-6,10-methylene-6H-pyrazino(2,3-h)benzodiazepine is selected from the group consisting of varenicline, compound 0765, and compound 0770.

[0373] 114. A method for identifying a ligand that selectively binds to a modified ligand-gated ion channel (LGIC), the method comprising:

[0374] providing one or more candidate ligands to the modified LGIC of item 1; and

[0375] Binding between the candidate ligand and the modified LGIC is detected, thereby identifying a ligand that selectively binds to the modified LGIC.

[0376] 115. The method of claim 114, wherein the modified LGIC is a chimeric LGIC comprising an LBD from a first LGIC and an IPD from a second LGIC.

[0377] 116. A method of detecting a modified ligand-gated ion channel (LGIC) comprising at least one modified LGIC subunit, the method comprising:

[0378] Providing one or more modified LGIC subunits of item 1;

[0379] providing a reagent that selectively binds to the modified LGIC; and

[0380] The binding between the modified LGIC and the agent that selectively binds to the modified LGIC is detected, thereby detecting the modified LGIC.

[0381] 117. The method of claim 116, wherein the agent that selectively binds to the modified LGIC comprises an antibody, a protein, or a small molecule.

[0382] 118. The method of claim 117, wherein the reagent that selectively binds to the modified LGIC comprises a detectable marker.

[0383] 119. The method of claim 118, wherein the detectable marker comprises a marker selected from the group consisting of a fluorescent marker, a radioactive marker, and a positron emitting marker.

[0384] 120. A mammalian cell comprising the modified LGIC of item 1.

[0385] 121. A nucleic acid expressing the modified LGIC subunit of item 1.

[0386] 122. A homomeric chimeric ligand-gated ion channel (LGIC), comprising chimeric LGIC subunits, each chimeric LGIC subunit comprising:

[0387] 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; and

[0388] glycine receptor ion pore domain;

[0389] wherein a ligand selected from the group consisting of tropisetron and granisetron selectively binds to the chimeric LGIC, and wherein the chimeric LGIC minimally binds acetylcholine (ACh).

[0390] 123. A homomeric chimeric ligand-gated ion channel (LGIC), comprising chimeric LGIC subunits, each chimeric LGIC subunit comprising:

[0391] an alpha 7 nicotinic acetylcholine receptor ligand binding domain having an L131G amino acid substitution and at least one of a Q79S amino acid substitution, a Q139L amino acid substitution, a Y217F amino acid substitution, an R27D amino acid substitution, and an E41R amino acid substitution; and

[0392] an ion pore domain selected from the group consisting of a glycine receptor ion pore domain and a serotonin 3 receptor ion pore domain;

[0393] wherein a ligand selected from varenicline and tropisetron selectively binds to the chimeric LGIC, and wherein the chimeric LGIC minimally binds acetylcholine (ACh).

[0394] 124. A method of treating a channelopathy in a mammal, the method comprising:

[0395] administering the LGIC of item 122 or item 123 to cells in said mammal; and

[0396] The ligand is administered to the mammal.

[0397] 125. A method for regulating cellular excitability in a mammal, the method comprising:

[0398] administering the LGIC of item 122 or item 123 to cells in said mammal; and

[0399] The ligand is administered to the mammal.

[0400] 126. A method of modulating cellular activity in a mammal, the method comprising:

[0401] administering the LGIC of item 122 or item 123 to cells in said mammal; and

[0402] The ligand is administered to the mammal.

Claims

1. A nucleic acid encoding a modified α7-nicotinic acetylcholine receptor (α7-nAChR) ligand binding domain (LBD), wherein the modified α7-nAChR LBD is composed of composition: Amino acids of SEQ ID NO:2 having (a) L131G amino acid substitution; (b) L131G amino acid substitution and Q139L amino acid substitution; (c) L131G amino acid substitution and Y217F amino acid substitution; or (d) L131G amino acid substitution, Q139L amino acid substitution and Y217F amino acid substitution.

2. A nucleic acid encoding at least one modified ligand-gated ion channel (LGIC) subunit comprising the modified α7-nAChR LBD and ion pore domain (IPD) of claim 1.

3. A nucleic acid encoding a modified ligand-gated ion channel (LGIC) comprising at least one modified LGIC subunit of claim 2.

4. The nucleic acid of claim 2 or 3, wherein the ion pore domain (IPD) is a serotonin 3 receptor (5HT3) IPD.

5. The nucleic acid of claim 2 or 3, wherein the ion pore domain (IPD) is a gamma-aminobutyric acid (GABA) receptor IPD.

6. The nucleic acid of claim 2 or 3, wherein the ion pore domain (IPD) is an α7-nicotinic acetylcholine receptor (α7-nAChR) IPD.

7. The nucleic acid of any one of claims 1-6, wherein the modified α7-nAChR LBD has a L131G amino acid substitution.

8. The nucleic acid of any one of claims 1-6, wherein the modified α7-nAChR LBD has an L131G amino acid substitution and a Q139L amino acid substitution.

9. The nucleic acid of any one of claims 1-6, wherein the modified α7-nAChR LBD has an L131G amino acid substitution and a Y217F amino acid substitution.

10. The nucleic acid of any one of claims 1-6, wherein the modified α7-nAChR LBD has an L131G amino acid substitution, a Q139L amino acid substitution, and a Y217F amino acid substitution.

11. The nucleic acid of any one of claims 1-10, wherein the modified α7-nAChR LBD further comprises an amino acid substitution at one or more amino acid residues selected from the group consisting of residues 175, 210, 216, and 219 as numbered in SEQ ID NO:

2.

12. The nucleic acid of claim 11, wherein the amino acid substitution is at (i) residue 175 of the α7-nAChR LBD, wherein the amino acid substitution is selected from the group consisting of G175A, G175F, G175H, G175K, G175M, G175R, G175S, and G175V; (ii) residue 210 of the α7-nAChR LBD, wherein the amino acid substitution is a Y210F substitution; (iii) residue 216 of the α7-nAChR LBD, wherein the amino acid substitution is a P216I substitution; (iv) residue 219 of the α7-nAChR LBD, wherein the amino acid substitution is a D219A substitution; or (v) any combination thereof.

13. The nucleic acid of any one of claims 3-12, wherein an exogenous ligand selectively binds to and activates the modified LGIC, and the modified LGIC has reduced activation potency with endogenous acetylcholine (ACh) relative to an LGIC lacking the modified LGIC subunit.

14. The nucleic acid of claim 13, wherein the modified LGIC has an EC50 of >20 μM for Ach.

15. The nucleic acid of claim 13, wherein the reduced activation potency with Ach is at least 3-fold reduced relative to a LGIC lacking the modified LGIC subunit.

16. The nucleic acid of any one of claims 13-15, wherein the exogenous 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-methylene-6H-pyrazino(2,3-h)benzaprozemide and 1,4-diazabicyclo[3.2.2]nonane.

17. The nucleic acid of claim 16, wherein the synthetic exogenous LGIC ligand is: (i) tropane, wherein the tropane is selected from the group consisting of tropisetron, pseudotropisetron, nortropisetron, Compound 723, Compound 725, Compound 737, and Compound 745; (ii) quinuclidine, wherein the quinuclidine is selected from the group consisting of PNU-282987, PHA-543613, compound 0456, compound 0434, compound 0436, compound 0354, compound 0353, compound 0295, compound 0296, compound 0536, compound 0676, and compound 702; (iii) 9-azabicyclo[3.3.1]nonane, and wherein the 9-azabicyclo[3.3.1]nonane is compound 536; (iv) 6,7,8,9-tetrahydro-6,10-methylene-6H-pyrazino(2,3-h)benzapine, wherein the 6,7,8,9-tetrahydro-6,10-methylene-6H-pyrazino(2,3-h)benzapine is selected from the group consisting of varenicline, Compound 765 and Compound 770; or (v) 1,4-diazabicyclo[3.2.2]nonane, 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 773, and Compound 774.

18. A mammalian cell comprising the nucleic acid of any one of claims 1 to 17.

19. A viral vector comprising the nucleic acid of any one of claims 1 to 17.

20. The viral vector of claim 19, wherein the viral vector is an adeno-associated viral vector.

21. Use of a nucleic acid according to any one of claims 3 to 17 or a viral vector according to claim 19 or 20 in the preparation of a medicament for treating a channelopathy in a mammal, wherein the medicament is used in a method, wherein the method include: i) administering the nucleic acid of any one of claims 3 to 17 or the viral vector of claim 19 or 20 to cells in said mammal; as well as ii) administering an exogenous LGIC ligand to said mammal.

22. The method of claim 21, wherein the channelopathy is selected from the group consisting of Bartter syndrome, Brugada syndrome, catecholamine-dependent ventricular tachycardia (CPVT), congenital hyperinsulinemia, cystic fibrosis, Dravet syndrome, paroxysmal ataxia, erythromelalgia, generalized epilepsy (e.g., with febrile seizures), familial hemiplegic migraine, fibromyalgia, hyperkalemic periodic paralysis, hypokalemic periodic paralysis, Lambert 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 deafness, paramyotonia congenita, retinitis pigmentosa, timothy syndrome, tinnitus, seizures, trigeminal neuralgia, and multiple sclerosis.

23. Use of a nucleic acid according to any one of claims 3 to 17 or a viral vector according to claim 19 or 20 in the preparation of a medicament for modulating ion transport across a mammalian cell membrane, wherein the medicament is used in a method wherein include: i) administering to the cell a nucleic acid according to any one of claims 3 to 17 or a viral vector according to claim 19 or 20; and ii) administering an exogenous LGIC ligand to said mammal.

24. The use of claim 23, wherein said modulation comprises activating ion transport.

25. The use of claim 23, wherein said modulation comprises inhibition of ion transport.

26. The use of claim 23, wherein the cells are selected from the group consisting of neurons, glial cells, muscle cells, stem cells, endocrine cells, and immune cells.

27. The use of claim 23, wherein administering the modified LGIC to the cell comprises administering in vivo.

28. Use of a nucleic acid according to any one of claims 3 to 17 or a viral vector according to claim 19 or 20 in the preparation of a medicament for regulating the excitability of cells in a mammal, wherein the medicament is used in a method, wherein the method include: i) administering the nucleic acid of any one of claims 3 to 17 or the viral vector of claim 19 or 20 to the mammalian cell; as well as ii) administering an exogenous LGIC ligand to said mammal.

29. The use of claim 28, wherein said modulation comprises increasing the excitability of said cells.

30. The use of claim 28, wherein said modulation comprises reducing the excitability of said cells.

31. The use of claim 28, wherein the cell is an excitable cell.

32. The use of claim 28, wherein the cell is selected from the group consisting of neurons, glial cells, muscle cells, stem cells, endocrine cells, and immune cells.

33. The use of claim 28, wherein administering the modified LGIC to the cell comprises administering in vivo.

34. Use of a nucleic acid according to any one of claims 3 to 17 or a viral vector according to claim 19 or 20 in the preparation of a medicament for modulating the activity of a cell in a mammal, wherein the medicament is used in a method, wherein the method include: i) administering to the cell a nucleic acid according to any one of claims 3 to 17 or a viral vector according to claim 19 or 20; and ii) administering an exogenous LGIC ligand to said mammal.

35. The use of claim 34, wherein said modulation comprises increasing the activity of said cell.

36. The use of claim 34, wherein said modulation comprises decreasing the activity of said cell.

37. The use of claim 34, wherein the activity is selected from the group consisting of ion transport, passive transport, excitation, inhibition, and exocytosis.

38. The use of claim 34, wherein the cells are selected from the group consisting of neurons, glial cells, muscle cells, stem cells, endocrine cells, and immune cells.

39. The use of claim 34, wherein administering the modified LGIC to a cell from the mammal comprises administering in vivo.

40. The use of claim 34, wherein administering the modified LGIC to cells from the mammal comprises ex vivo administration.

41. The use of any one of claims 21, 23, 28 and 34, wherein the exogenous LGIC ligand is a synthetic exogenous LGIC ligand.

42. The use of claim 41, wherein the synthetic exogenous LGIC ligand is: (i) tropane, wherein the tropane is selected from the group consisting of tropisetron, pseudotropisetron, nortropisetron, Compound 723, Compound 725, Compound 737, and Compound 745; (ii) quinuclidine, wherein the quinuclidine is selected from the group consisting of PNU-282987, PHA-543613, compound 0456, compound 0434, compound 0436, compound 0354, compound 0353, compound 0295, compound 0296, compound 0536, compound 0676, and compound 702; (iii) 9-azabicyclo[3.3.1]nonane, and wherein the 9-azabicyclo[3.3.1]nonane is compound 536; (iv) 6,7,8,9-tetrahydro-6,10-methylene-6H-pyrazino(2,3-h)benzapine, wherein the 6,7,8,9-tetrahydro-6,10-methylene-6H-pyrazino(2,3-h)benzapine is selected from the group consisting of varenicline, Compound 765 and Compound 770; or (v) 1,4-diazabicyclo[3.2.2]nonane, 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 773, and Compound 774.

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