NZ790090A - Modified ligand-gated ion channels and methods of use - Google Patents

Abstract

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

Description

WO 09832 MODIFIED LIGAND-GATED ION CHANNELS AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of US. Patent Application Serial No. 62/3 59,534, filed on July 7, 2016, and claims the benefit of US. Patent Application Serial No. 62/486,779, filed on April 18, 2017. The disclosures of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application. 1. Technical Field This document relates to materials and methods for controlling ligand gated ion channel (LGIC) activity. For example, this document provides modified LGICs including at least one LGIC t having a modified ligand binding domain (LBD) and/or a modified ion pore domain (IPD). Also ed are exogenous LGIC ligands that can bind to and activate the modified LGIC. In some cases, a modified LGIC and an exogenous ligand can be used to treat a mammal having a channelopathy (e.g., a neural channelopathy or a muscle channelopathy). In some cases, a modified LGIC and an exogenous LGIC ligand can be used to modulate (e.g., activate or inhibit) ion transport across the membrane of a cell of a mammal. In some cases, a modified LGIC and an exogenous LGIC ligand can be used to modulate (e.g., increase or decrease) the excitability of a cell in a mammal. 2. Background Information Ion channels mediate ionic flux in cells, which profoundly s their biological function. A prominent instance of this is in neurons, where ion channels l electrical signaling within between s to influence physiology, sensation, behavior, mood, and cognition.

Different LGICs have distinct ligand binding properties as well as specific ion tance 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 ors s) bind the endogenous ligand acetylcholine (ACh), which tes conductances for cations and typically depolarizes cells, thereby increasing cellular excitability. In contrast, the glycine receptor (GlyR) binds the endogenous ligand glycine, which tes chloride anion conductance and typically reduces the excitability of cells by hyperpolarization and/or by an electrical shunt of the cellular membrane resistance.

SUMMARY Levels of endogenous LGIC agonists such as ACh are not readily controlled.

This document provides materials and methods for controlling LGIC activity (e.g., increasing the sensitivity of LGICs to exogenous ligands and/or reducing sensitivity to endogenous ligands such as ACh). For example, this document provides modified LGICs including at least one modified LGIC subunit having a LED and an IPD, and having at least one modified amino acid (e.g., an amino acid substitution). Also provided are ous LGIC ligands that can bind to and activate the modified LGIC. In some cases, a modified LGIC and an exogenous ligand can be used to treat a mammal having a channelopathy (e.g., a neural channelopathy or a muscle channelopathy). In some cases, a modified LGIC and an exogenous LGIC ligand can be used to modulate (e.g., activate or inhibit) ion transport across the membrane of a cell of a . In some cases, a modified LGIC and an exogenous LGIC ligand can be used to modulate (e.g., increase or decrease) the excitability of a cell in a mammal.

Having the ability to l LGIC activity provides a unique and unrealized unity to e l of ion transport in cells. For example, modified LGICs having increased sensitivity for one or more ous LGIC ligands can be used to provide temporal and spatial control of ion transport and/or cellular excitability based on delivery of the ous LGIC ligand. For example, modified LGICs with reduced sensitivity for endogenous LGIC ligands t unwanted activation of modified LGICs and allow for selective control over the modified LGIC by exogenous ligands. Further, ous LGIC s having increased potency for a modified LGIC improve ivity of targeting of the modified LGIC over endogenous ion channels. Thus, the modified LCIGs and exogenous LGIC ligands provided herein are useful to e a therapeutic effect while reducing side effects from the small molecules on unintended targets.

As described herein, one or more mutations in a modified LGIC can enhance potency for exogenous LGIC ligands. Mutation of the 0L7 LBD of d7-GlyR at residue L131 (e. g., substituting Leu with Gly or Ala) increased potency for varenicline (16-fold) and tropisetron (36-fold) while reducing ACh potency (-6.4-fold) relative to 0L7-GlyR. Mutation of 0L7 LBD of 0L7-GlyR at residue G175 (e.g., G175K) or P216 (e.g., P2161) enhanced potency for ACh, nicotine, tropisetron, varenicline, as well as other lidine and tropane agonists.

Combining the mutation at residue G175K with mutations that reduce y of the endogenous agonist ACh (e.g. Y115F) produced 0t7-GlyR Y115F G175K with increased y for tropisetron (55-fold) and reduced potency from ACh (fold). In on, combining mutations in the 0L7 LBD at residues 77 (e.g., substituting Trp with Phe or Tyr) and/or 79 (e.g., substituting Gln with Gly, Ala, or Ser) and/or 131 (e. g., substituting Leu with Gly or Ala) and/or141 (e. g., substituting Leu with Phe or Pro) in these chimeric channels with potency enhancing mutations at residues G175 (e.g., G175K) or P216 (e.g., P2161) se potency for distinct ligands and/or reduce ACh potency. For example, a chimeric 0L7-GlyR LGIC with a 0L7 nAChR LBD (0L7 LBD) having a on at e 79 (e.g., substituting Gln with Gly), a on at residue 115 (e.g., substituting Tyr with Phe), and a mutation at residue 175 (e.g., substituting Gly with Lys) has greater than lOO-fold increased sensitivity to an exogenous tropane LGIC ligand compound 723 (a tropane), and reduced ACh sensitivity (fold) relative to the unmodified chimeric 0L7-GlyR LGIC. Furthermore, a modified LGIC including at least one chimeric LGIC subunit having an 0L7 nAChR LBD (0L7 LBD) having a mutation at residue 79 (e.g., substituting Gln with Ala, Gly, or Ser) and a GlyR IPD having a mutation at residue 298 (e.g., tuting Ala with Gly) has nearly 20- fold increased sensitivity for an exogenous LGIC ligand, such as a quinuclidine or a tropane.

Additional mutations at residue 27 (e.g., substituting Arg with Asp) and 41 (e.g., substituting Glu with Arg) of the 0L7 LBD reduced the association of the modified chimeric LGIC with an unmodified ion channels. Additional mutations at residue 115 (e.g., tuting Tyr with Phe), 139 (e.g., substituting Gln with Gly or Leu), 210 (e.g., substituting Tyr with Phe) 217 (e.g., substituting Tyr with Phe), and/or 219 (e.g., substituting Asp with Ala) of the 0L7 LBD reduced sensitivity of the chimeric LGIC to the endogenous ligand ACh. These chimeric LGICs allow for highly selective control over cellular function in cells of a mammal while minimizing cross-reactivity with nous ing s in the mammal.

In general, one aspect of this document features a modified LGIC having at least one modified LGIC subunit which includes a LBD having an amino acid modification, and an IPD, where an exogenous LGIC ligand activates the modified LGIC. The d LGIC can be a chimeric LGIC having a LBD from a first LGIC and an IPD from a second LGIC.

The LBD can be an alpha7 nicotinic choline receptor (0L7-nAChR) LBD. The modified LGIC of claim 3, wherein the at least one modified amino acid in the 0L7-IlAChR 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 0L7-11AChR LBD. The amino acid tution can be at residue 79 of the 0L7 LBD, and the amino acid substitution can be Q79A, Q79G or Q79S. For example, the amino acid substitution at residue 79 of the 0L7 LBD can be Q79G. The IPD can be a serotonin 3 receptor (5HT3) IPD, a glycine receptor (GlyR) IPD, a gamma-aminobutyric acid (GABA) receptor IPD, or an 0L7-11AChR IPD. The IPD can be a GlyR IPD, and the GlyR IPD can include an amino acid substitution at residue 298 (e.g., a A298G substitution) of the ic LGIC. The IPD can be a GABA IPD, and the GABA IPD can include an amino acid substitution at residue 298 (e.g., a W298A substitution) of the modified LGIC. The modified LGIC can be a chimeric LGIC including an 0L7 LBD haVing a Q79G amino acid substitution, and a GlyR IPD haVing a A298G amino acid substitution. The exogenous LGIC ligand can be a synthetic exogenous LGIC ligand selected from the group consisting of a quinuclidine, a tropane, a 9-azabicyclo[3.3.1]nonane, a 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3-h)benzazepine, and a 1,4- diazabicyclo[3.2.2]nonane. When the synthetic exogenous LGIC ligand is a tropane, the e can be etron, pseudo-tropisetron, pisetron, compound 723, compound 725, compound 737, or compound 745. When the synthetic exogenous LGIC ligand is a quinuclidine, the quinuclidine can be PNU-282987, FHA-543613, compound 0456, compound 0434, compound 0436, compound 0354, compound 0353, compound 0295, compound 0296, compound 0536, compound 0676, or compound 702. When the synthetic exogenous LGIC ligand is a 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino(2,3- h)benzazepine, the ligand can be compound 765 or compound 770. When the synthetic exogenous LGIC ligand is a azabicyclo[3.2.2]nonane, the ligand can be compound 773 or compound 774. In some cases, the LBD can be an 0L7 LBD, and the 0L7 LBD can also include at least one modified amino acid that confers ive binding to another 0L7 LBD haVing the at least one modified amino acid over binding to an unmodified LGIC. The unmodified LGIC can be an endogenous LGIC (e.g., an endogenous 0L7-IlAChR). The at least one modified amino acid in the 0L7 LBD that s reduced binding to the unmodified LGIC can include an amino acid substitution at residue 27 (e.g., a R27D tution) and/or residue 41 (e. g., an E41R substitution). In some cases, the IPD can be a 5HT3 IPD, and the 5HT3 IPD can include at least one modified amino acid that s increased ion conductance to the modified LGIC. The at least one modified amino acid in the 5HT3 IPD that confers increased ion conductance to the modified LGIC can e an amino acid substitution at an amino acid e at e 425 (e.g., a R425Q substitution), 429 (e.g., a R429D substitution), and/or 433 (e.g., a R433A substitution).

In r aspect, this document features a modified LGIC having at least one modified LGIC subunit ing a LBD having at least one modified amino acid, and an IPD, where the at least one modified amino acid in the LBD reduces binding with an endogenous LGIC ligand. The modified LGIC can be a chimeric LGIC having a LBD from a first LGIC and an IPD from a second LGIC. The endogenous LGIC ligand can be ACh. The modified LGIC can have an ECSO of greater than 20 uM for Ach. The at least one modified amino acid can include an amino acid substitution at residue 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 tution can be a Y115F substitution. When the at least one modified amino acid includes an amino acid substitution at e 139, the amino acid substitution can be a Q139G or a Q139L substitution. When the at least one modified amino acid includes an amino acid substitution at residue 210, the amino acid substitution can be a Y210F substitution. When the at least one modified amino acid includes an amino acid substitution at residue 217, the amino acid substitution can be a Y217F substitution. When the at least one modified amino acid includes an amino acid substitution at residue 219, the amino acid substitution can be a D219A substitution.

In another aspect, this document features a ligand having increased potency for a modified ligand gated ion channel (LGIC), wherein the ligand comprises Formula I: Gain} «’1', "Carri \/~}('3 a", 1 ‘ " RV "Y \ r?" X3W£< where each of X1, X2, and X3 can independently be CH, CH2, 0, NH, or We, where each n can independently be 0 or 1, where Y = O or S, where A = an aromatic substituent, and where R: H or nylmethylene. The ic substituent can be lH-indole, 4- (trifluoromethyl) benzene, 2,5-dimethoxy benzene, 4-chloroaniline, aniline, 5- (trifluoromethyl) pyridinyl, 6-(trifluoromethyl) nicotinic, or 4-chloro-benzene.

In some cases, a LGIC ligand can be a quinuclidine and can have a structure shown in Formula II: rKL?" xii-W» A t N N? j); \ Y where X3 = O, NH, or CH2, where Y = O or S, where A = an aromatic substituent, and where R = H or pyridinylmethylene. The aromatic substituent can be lH-indole, 4-(trifluoromethyl) benzene, 4-chloro benzene, methoxy benzene, 4-(trifluoromethyl) benzene, 4- chloroaniline, aniline, 5-(trifluoromethyl) pyridinyl, fluoromethyl) nicotinic, 3- chlorofluoro benzene, or ole. The quinuclidine can be PNU-282987, PHA-543613, compound 0456, compound 0434, compound 0436, compound 0354, compound 0353, compound 0295, compound 0296, compound 0536, compound 0676, or compound 702.

In some cases, a LGIC ligand can be a tropane and can have a structure shown in Formula III: gLL\I, \\ ’9’ X3"A\ where X2 = NH or NMe, where X3 = O, NH, or CH2, where Y = O or S, and where A = an aromatic substituent. The aromatic tuent can be lH-indole, 7-methoxy-lH-indole, 7- -lH-indole, 5-chloro-1H-indole, or lH-indazole. The tropane can be tropisetron, pseudo-tropisetron, nortropisetron, compound 723, compound 725, compound 737, or compound 745.

In some cases, a LGIC ligand can be a 9-azabicyclo[3.3. l]nonane and can have a structure shown in Formula IV: V‘v’ 5N x "x , \\i .\ x»l where X1 can be CH, X2 can be NH or We, X3 can be 0, NH, or CH, Y can be 0 or S, and A can be an aromatic substituent. The aromatic substituent can be 4-chloro-benzene. The 9- azabicyclo[3.3.l]nonane can be compound 0536.

In another aspect, this document features a ligand having increased potency for a modified ligand gated ion channel , where the ligand can be a 6,7,8,9-tetrahydro-6,10- methano-6H-pyrazino(2,3-h)benzazepine and have a structure shown in Formula V: HNg: NA where R can be H or CH3, and where A can be H or an aromatic substituent. The 6,7,8,9- tetrahydro-6, l0-methano-6H-pyrazino(2,3-h)benzazepine can be varenicline, compound 0765, or nd 0770.

In r aspect, this document features a ligand haVing increased potency for a modified ligand gated ion channel (LGIC), where the ligand can be a 1,4- diazabicyclo[3.2.2]nonane and can have a ure shown in Formula VI: MN where R can be H, F, or N02. The l,4-diazabicyclo[3.2.2]nonane can be 3-(l,4- diazabicyclo[3.2.2]nonanyl)dibenzo[b,d]thiophene 5,5-dioxide, compound 0773, or compound 0774.

In another aspect, this document es methods of treating a channelopathy in a mammal. The methods include, or consist essentially of, administering to a cell in the mammal a d LGIC, where an exogenous LGIC ligand selectively binds the modified LGIC. The modified LGIC has at least one modified LGIC subunit including a LBD including at least one modified amino acid, and an IPD, and administering the exogenous ligand to the mammal. The channelopathy can be Bartter syndrome, a syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT), congenital hyperinsulinism, cystic fibrosis, Dravet syndrome, episodic ataxia, erythromelalgia, generalized epilepsy (e.g., with febrile seizures), familial hemiplegic ne, fibromyalgia, hyperkalemic periodic paralysis, hypokalemic ic paralysis, Lambert-Eaton myasthenic syndrome, long QT syndrome (e.g., Romano-Ward syndrome), short QT syndrome, malignant hyperthermia, mucolipidosis type IV, myasthenia gravis, myotonia congenital, neuromyelitis optica, neuromyotonia, nonsyndromic ss, paramyotonia congenital, retinitis pigmentosa, timothy syndrome, tinnitus, seizure, trigeminal gia, or multiple sclerosis.

In another aspect, this document features methods of modulating ion transport across a cell membrane of a mammal. The methods include, or t essentially of, administering to the cell a modified LGIC, where an exogenous LGIC ligand selectively binds the d LGIC. The modified LGIC has at least one modified LGIC subunit including a LBD including at least one modified amino acid, and an IPD, and administering the exogenous ligand to the mammal. The modulating can include ting or inhibiting ion transport.

The cell can be a neuron, a glial cell, a myocyte, a stem cell, an endocrine cell, or an immune cell. The administering the modified LGIC to the cell can be an in vivo administration or an ex vivo administration. The administering the modified LGIC to the cell can include administering a nucleic acid ng the modified LGIC.

In another aspect, this document features s of modulating the excitability of a cell in a mammal. The methods include, or consist essentially of, administering to the cell from the mammal a modified LGIC, where an exogenous LGIC ligand selectively binds the modified LGIC. The modified LGIC has at least one modified LGIC subunit including a LBD including at least one modified amino acid, and an IPD, and administering the exogenous ligand to the mammal. The modulating can include increasing the excitability of the cell or decreasing the excitability of the cell. The cell can be an excitable cell. The cell can be a neuron, a glial cell, a myocyte, a stem cell, an endocrine cell, or an immune cell.

The administering the modified LGIC to the cell can be an in vivo administration or an ex vivo administration. The administering the modified LGIC to the cell can include stering a nucleic acid encoding the modified LGIC.

In another , this nt features s of modulating the activity of a cell in a . The methods include, or consist essentially of, administering to the cell a modified LGIC, where an ous LGIC ligand selectively binds the modified LGIC. The modified LGIC has at least one modified LGIC subunit ing a LED including at least one modified amino acid, and an IPD, and administering the exogenous ligand to the mammal. The modulating can include increasing the ty of the cell or decreasing the activity of the cell. The activity can be ion transport, passive transport, excitation, inhibition, or osis. The cell can be a neuron, a glial cell, a myocyte, a stem cell, an endocrine cell, or an immune cell. The stering the modified LGIC to the cell can be an in vivo administration or an ex vivo administration. The administering the modified LGIC to the cell can e administering a nucleic acid (e.g., via a viral vector such as an adeno-associated virus, a herpes simplex virus, or a lentivirus) encoding the modified LGIC.

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

In another aspect, this document features a method for detecting a modified LGIC.

The method includes, or consists essentially of, providing one or more modified LGIC subunits described herein, providing an agent that selectively binds the modified LGIC, and detecting g between the modified LGIC and the agent that selectively binds the modified LGIC, thereby detecting the modified LGIC. The agent that selectively binds the modified LGIC comprises can be antibody, a protein (e.g., bungarotoxin), or a small molecule (e.g., a positron emission aphy (PET) ligand). The agent that selectively binds the modified LGIC can e a detectable label (e.g., a fluorescent label, a ctive label, or a on emitting label).

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

The details of one or more embodiments of the ion are set forth in the accompanying drawings and the description below. Other features, s, and ages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS Figure 1 shows exemplary amino acid sequences of ic LGICs. Mutation of amino acid residue 77 (e.g., W77F or W77Y) resulted in sensitivity to granisetron and tropisetron. Mutation of amino acid residue 79 (e.g., Q79G) was most effective for several agonists. Mutations of amino acid residue 131 (e.g., L131G, L131A, L131M, or L131N) altered sensitivity to varenicline, tropisetron, granisetron, and ACh. Potency was considerably enhanced when LBD mutations were combined with mutation at amino acid residue 298 in the GlyR or GABAC IPD. Potency was also enhanced when 0L7 nAChR LBD mutations were combined with mutation at amino acid residue G175 and P216. A) An amino acid sequence of 0L7-5HT3 chimeric or (SEQ ID NO:6) including a human 0L7 nAChR LBD (SEQ ID NO: 1) and a murine 5HT3 IPD (SEQ ID NO:3) components. B) An amino acid sequence of 0L7-GlyR chimeric receptor (SEQ ID NO:7), including a human 0L7 nAChR LBD (SEQ ID NO:2) and a human GlyR IPD (SEQ ID NO:5) components. C) An amino acid sequence of 0L7-5HT3 chimeric receptor (SEQ ID NO:8) including human 0L7 nAChR LBD (SEQ ID NO: 1) and a human 5HT3 IPD (SEQ ID NO:4) components. D) An amino acid sequence of 0L7- GABAc chimeric or (SEQ ID NO: 10) including a human 0L7 nAChR LBD (SEQ ID NO:2) and a human GABAc IPD (SEQ ID NO:9) components. E) An amino acid sequence of rat nAChR sequence (SEQ ID NO: 12).

Figure 2 shows ECSOs for tropisetron against a 0L7-5HT3 chimeric LGIC and ts of the chimeric LGIC with LBD mutations at positions noted in Figure 1. Multiple mutations at Gln79 showed similar or improved potency relative to the unmodified d7-5HT3 channel (arrows).

Figure 3 shows the relative potency of known nAChR agonists for d7-5HT3 chimeric LGICs. A) A graph of ECSOs normalized to the fied 0L7-5HT3 chimeric channel (log scale). >"P Figure 4 shows the relative potency of known nAChR agonists for d7-GlyR chimeric LGICs. A) A graph of ECSOs for Q79 LBD mutants ized to the unmodified 0L7-GlyR chimeric channel (log scale). B) A graph of ECSOs for A298G IPD on normalized to the unmodified 0L7-GlyR chimeric l (log scale). C) A graph of ECSOs for 0L7- GlyRAZ98G ized to the unmodified 0L7-GlyR chimeric channel and compared to the double mutant channel 0L7Q79G-GlyRA298G (log scale). >"P Figure 5 shows schematic structures of LGIC agonists with substitution patterns most compatible with y enhancement for 0L7Q79G-5HT3 and 0t7Q79G-GlyRA298G. A) A generalized structure showing attributes ated with enhanced potency. B) Specific pharmacophores represented in (A) are quinuclidine, tropane, and 9-azabicyclo[3.3.l]nonane core structures. C) Exemplary synthetic molecules that show high potency for 0L7Q79G- QSG, U7Q79G’Y115F’G175K-GlYR, U7W77F’Q79G’G175K-GlYR.

Figure 6 shows mutations that reduce association of chimeric LCIG 0L7 nAChR LBDs with unmodified LBDs. A) Charge reversal schematic potential configurations of transfecting two epitope tagged (HA and V5) constructs encoding T3 (top) or two constructs encoding 0L7-5HT3-HA and d7R21D’E4lR-5HT3 -V5 where association between the two different epitope tagged subunits would be unfavored due to charge reversal mutations at the subunit interfaces. B) Whole cell recordings in HEK cells expressing 0L7R21D’E41R-5HT3 with a V5 epitope tag shows potent responses to PNU-282987. C) ation of 0L7-5HT3 LGICs with HA and V5 epitope tags in HEK cells was probed by HA immunoprecipitation (left) or total lysate isolation followed by western ng with either anti-HA (top) or anti- V5 antibodies (bottom). In cells ressing channels with the HA and V5 epitopes, anti- HA IP ed by anti-V5 immunoblotting shows the unoprecipation of unmodified channels of each type, but charge reversal mutations in the LBD 0t7R21D’E41R-5HT3-V5 was not immunoprecipitated. MW of 0L7-5HT3 is ~48 kD (arrow).

Figure 7 shows that chimeric LGICs can be lled using an exogenous ligand.

Cortical neurons from a mouse brain transduced with 0L7Q79G-GlyRAZ98G ic LGIC via adeno-associated virus (AAV) vectors fires action potentials in response to 40 pA current injection (PRE) that are potently suppressed by 30 nM tropisetron. After washout (WASH) of tropisetron, neuron firing is restored.

Figure 8 shows activity of agonists on chimeric LGICs with a G175K mutation. A) A graph of ECSOs for Q79G G175K LBD mutants against known agonists normalized to the unmodified 0L7-GlyR chimeric channel (log scale). B) A graph of ECSOs for ACh and tropisetron for channels with mutations in 0L7-GlyR chimeric LGICs. Mutations that result in channels with high potency for etron and low potency for the endogenous , acetylcholine (ACh) are l (grey shading). Unmod.: fied d7-GlyR chimeric LGIC. C) Action potentials of cortical neurons from a mouse brain transduced with d7Q79G’Y115F’G175K-GlyR chimeric LGIC. Neurons fire in response to current injection (PRE) and are potently suppressed by 100 nM tropisetron. After t (WASH) of tropisetron, neuron firing is restored.

Figure 9 shows activity of agonists on chimeric LGICs with a L131G mutation. A) A graph of ECSOs for L131 LBD mutants t known agonists normalized to the unmodified 0L7-GlyR chimeric channel (log scale). B) A graph of ECSOs for ACh and tropisetron for channels with mutations in 0L7L131G-GlyR chimeric LGICs. C) A graphs g mutations that result in channels with high potency for varenicline and low potency for the endogenous , acetylcholine (ACh) are optimal (grey shading). Unmod.: unmodified 0L7-GlyR chimeric LGIC. D) Action potentials of a cortical neuron from a mouse brain transduced with 0L7L131G’Q139L’Y217F—G1yR chimeric LGIC. Neuron fires in response to current injection (PRE) and are potently suppressed by 10 nM varenicline, even with >6-fold greater injected current. After washout (WASH) of tropisetron, neuron firing is restored.

Figure 10 shows chemical structures of LGIC ts. A) Chemical structures of LGIC agonists with substitution patterns most compatible with y enhancement for d7Q79G’Y115F’G175K-GlyR. B) Chemical structures of LGIC agonists with tution patterns most compatible with y enhancement for 0L7L131G’Q139L’Y217F-G1yR or d7L131G’Q139L’Y217F- 5HT3 HC.

DETAILED DESCRIPTION This document provides modified LGICs and methods of using them. For example, this document provides modified LGICs including at least one modified LGIC subunit having a LBD and an IPD, and having at least one modified amino acid (e.g., an amino acid substitution). In some cases, a modified LGIC can be a chimeric LGIC. For example, a chimeric LGIC can include a LBD from a first LGIC and an IPD from a second LGIC. In some cases, the modified amino acid can confer cological selectivity to the modified LGIC. For e, the modified amino acid can confer the modified LGIC with selective binding of an exogenous LGIC ligand. For example, the modified amino acid can confer the d LGIC with reduced (minimized or eliminated) binding of an unmodified LGIC subunit (an LGIC subunit lacking the modification and/or an endogenous LGIC subunit).

For example, the modified amino acid can confer the modified LGIC with reduced (minimized or eliminated) g of an endogenous LGIC ligand.

Modified LGICs ed herein can be used, for example, in methods for treating channelopathies (e.g., a neural channelopathy or a muscle channelopathy). For example, a modified LGIC, and an exogenous LGIC ligand that can bind to and activate the modified LGIC, can be used to treat a mammal having a channelopathy. In some cases, a modified LGIC and an exogenous LGIC ligand can be used to modulate (e.g., activate or inhibit) ion transport across the membrane of a cell of a mammal. In some cases, a modified LGIC and an exogenous LGIC ligand can be used to modulate (e.g., se or decrease) the excitability of a cell in a .

ModifiedLGICS As used herein a "modified" LGIC is an LGIC that includes at least one LGIC subunit. A modified LGIC subunit can include at least one modified amino acid (e.g., an amino acid substitution) in the LED and/or at least one modified amino acid (e.g., an amino acid substitution) in the IPD. A modified LGIC subunit described herein can be a modification of an LGIC from any appropriate species (e.g., human, rat, mouse, dog, cat, horse, cow, goat, pig, or monkey). In some cases, a modified LGIC can include at least one chimeric LGIC subunit having a non-naturally occurring ation of a LED from a first LGIC and an IPD from a second LGIC.

A modified LGIC can be a homomeric (e.g., having any number of the same modified LGIC subunits) or heteromeric (e.g., having at least one modified LGIC subunit and any number of different LGIC subunits). In some cases, a modified LGIC described herein can be a homomeric modified LGIC. A modified LGIC described herein can include any suitable number of modified LGIC subunits. In some cases, a d LGIC can be a trimer, a tetramer, a pentamer, or a hexamer. For example, a modified LGIC described herein can be a A modified LGIC subunit bed herein can be a modification of any appropriate LGIC. The LGIC can t anions, cations, or both through a cellular membrane in response to the binding of a ligand. For example, the LGIC can transport sodium (Na+), potassium (K+), calcium (Ca2+), and/or chloride (Cl—) ions through a cellular membrane in response to the binding of a ligand. Examples of LGICs include, without limitation, Cys- loop receptors (e.g., AChR such as a nAChR (e.g., a muscle-type nAChR or a neuronal-type nAChR), gamma-aminobutyric acid (GABA, such as GABAA and GABAA-p (also referred to as GABAc) receptors, GlyR, GluCl receptors, and 5HT3 receptors), opic glutamate receptors (iGluR, such as AMPA receptors, kainate receptors, NMDA receptors, and delta receptors), ATP-gated channels (e.g., P2X), and phosphatidylinositol 4,5-bisphosphate (PIP2)—gated channels. In cases where a modified LGIC described herein is a chimeric LGIC, the chimeric LGIC can include a LBD ed from any riate LGIC and an W IPD selected from any appropriate LGIC. In cases where a LGIC includes multiple ent subunits (for example, a neuronal-type nAChR includes d4, [32, and 0L7 ts), the LBD and/or IPD can be selected from any of the subunits. For example, a LBD from a nAChR can be a 0L7 LBD. A representative rat 0L7 nAChR amino acid sequence (including both a LBD and an IPD) is as follows.

SEQ ID NO:12 MGGGQGG"WLALAAALLHVSLQG?FQRRLY SEQ ID NO:1 MQCSPGGVWLALAASLLHVSLQG?FQRKLY{ELVKWYNPLEQPVANDSQPLTVYFSLSLLQ: MDVDEKNQVLTTN"WLQMSWTDHYLQWNVSEYPGV{TVQEPDGQ W In some cases, a 5HT3 IPD can be have at least 75 percent sequence identity (e.g., at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 97% or at least 99% sequence identity) to SEQ ID NO:3 of SEQ ID N04.

SEQ ID NO:3 RRRBLEYAVSLLLBS ETMVVD VGECLBBDSGLRVSEKHTLLLGYSVEL VSDTLPAT GTPL GVYEVVCMALLV SLAflT b'VRLV PVPDWLRILVLDR AW LCLGfiQP ATFQAN SEQ ID NO:5 MGYYL QMY PSLL V LSW SEW NMDAAPARVGLG:TTVLTMTTQSSGSRASLPKVSYVK A D WMAVCLLFVFSALLEYAAVNFVSQQH{ELLRFRRKQRHHKfiDfiAGfiGQENESAYGMGP DG:SV SEQ ID NO:9 LLQTYFPATLMVMLSWVS FW I DRRAVPARVPT .G " T TVTITMS T TGVNASMBRVSY KAVD YLWVS FVFVFT.SVTFYAAVNYTE‘TVQ *ZRK *ZQKTR CTSGLPPPRTAMLDGNYSDGEVWD LDNYMB*ZNG*ZKPDRWMVQLTLASERSSPQQKSQQSSYVSMR DTHA DKYS? Jr'PAAY T.J:' NT. YWS b'S In calculating percent sequence ty, two sequences are aligned and the number of identical matches of amino acid es between the two ces is determined. The number of identical matches is divided by the length of the aligned region (i.e., the number of aligned amino acid residues) and multiplied by 100 to arrive at a percent sequence identity value. It will be appreciated that the length of the aligned region can be a portion of one or both sequences up to the ength size of the shortest sequence. It also will be appreciated that a single sequence can align with more than one other sequence and hence, can have different percent sequence identity values over each d . The alignment of two or more ces to determine percent sequence identity can be performed using the computer program ClustalW and default parameters, which calculates the best match between a query and one or more subject sequences, and aligns them so that identities, similarities and differences can be determined. See, e. g., Chenna et al., 2003, Nucleic Acids Res, 31(13):3497—500.

In cases where a modified LGIC subunit described herein is a chimeric LGIC subunit, the chimeric LGIC subunit can include a LED and IPD from the same species or a LED and IPD from different species. In some cases, a chimeric LGIC subunit can include a LED from a human LGIC protein and an IPD from a human LGIC protein. For example, a chimeric LGIC subunit can include a human 0L7 LED and a human GlyR IPD. In some cases, a chimeric LGIC subunit can include a LED from a human LGIC protein and an IPD from a murine LGIC protein. For example, a chimeric LGIC subunit can include a human 0L7 LED and a murine 5HT3 IPD.

In cases where a modified LGIC subunit described herein is a chimeric LGIC subunit, the chimeric LGIC subunit can e varied fusion points connecting the LED and the IPD such that the number of amino acids in a LBD may vary when the LBD is fused with different IPDs to form a chimeric channel subunit. For example, the length of an 0L7 nAChR LBD used to form a chimeric LGIC subunit with a 5HTS IPD is different from the length of an 0L7 nAChR LBD used to form a chimeric LGIC subunit with a GlyR IPD re, for example, Figures 1A and 1C to Figure 1B).

A modified LGIC t described herein can include a LBD having at least one modified amino acid and/or an IPD having at least one modified amino acid. For example, a modified LGIC subunit described herein can include a 0L7 LBD having at least 75 percent sequence ty to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 11, or SEQ ID NO: 12, and an amino acid substitution at amino acid residue 27, 41, 77, 79, 131, 139, 141, 175, 210, 216, 217, and/or 219. For example, a modified LGIC subunit described herein can include a GlyR IPD having at least 75 percent sequence identity to a sequence set forth in SEQ ID N05, and an amino acid substitution at amino acid e 298 of an d7-GlyR chimeric receptor (e.g., SEQ ID NO:7). For example, a modified LGIC subunit described herein can include a GABAc IPD having at least 75 percent sequence identity to SEQ ID N09, and an amino acid tution at amino acid residue 298 of an 0L7-GABAC chimeric receptor (e.g., SEQ ID NO: 10). In some cases, a modified LGIC subunit described herein can include more than one (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more) amino acid modifications. The modification can be an amino acid substitution. In some cases, the d amino acid can confer pharmacological selectivity to the modified LGIC. For example, the modified amino acid can confer the modified LGIC with selective binding of an exogenous LGIC . For example, the modified amino acid can confer the modified LGIC with reduced (minimized or eliminated) binding of an unmodified LGIC subunit (an LGIC subunit lacking the modification and/or an endogenous LGIC subunit). For example, the modified amino acid can confer the modified LGIC with reduced (minimized or eliminated) g of an endogenous LGIC ligand.

In some aspects, a modified LGIC t described herein can include at least one modified amino acid that confers the modified LGIC with selective binding (e.g., enhanced binding or sed potency) with an exogenous LGIC . The binding with an exogenous LGIC ligand can be selective over the binding with an endogenous LGIC .

A modified LGIC subunit with selective binding with an exogenous LGIC ligand can include any appropriate LDB (e.g., a 0L7 LBD). In some aspects, the modified LGIC subunit can include a 0L7 LBD set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO: 12, and the amino acid modification can be a tution at amino acid residue 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 NOzll, or SEQ ID NO: 12 can be substituted with a hydrophobic amino acid e such as phenylalanine (e.g., W77F), tyrosine (e.g., W77Y), or methionine (e.g., W77M). For example, a d LGIC subunit described herein can include a 0L7 LBD set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO: 12 and having a W77F substitution. In 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, a modified LGIC subunit described herein can include a 0L7 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: 1 1, 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: 1 1, or SEQ ID NO: 12 can be substituted with an amino acid residue such as lysine (e.g., G175K), alanine (e.g., G175A), phenyalanine (e.g., G175F), histidine (e.g., G175H), methionine (e.g., G175m), arginine (e.g., G175R), serine (e.g., G17SS), 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: 1 1, or SEQ ID NO: 12 can be tuted with an amino acid residue such as isoleucine (e.g., P2161). A modified LGIC subunit with selective binding with an exogenous LGIC ligand can include any appropriate IPD (e.g., a GlyR IPD or a GABAA-p IPD). In some aspects, the modified LGIC subunit can include a GlyR IPD set forth in SEQ ID N05, and the amino acid modification can be a substitution at amino acid e 298 of an d7-GlyR chimeric receptor (e.g., SEQ ID NO:7). In some cases, the alanine at amino acid residue 298 of SEQ ID NO:7 can be tuted with an amino acid residue such as glycine (e.g., A298G). In some aspects, the d LGIC subunit can include the a GABAA-p IPD set forth in SEQ ID N09, and the amino acid modification can be a substitution at amino acid residue 298 of an 0L7-GABAA-p ic or (e.g., 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 (e.g., W298A).

In some cases, a d LGIC subunit described herein can include more than one (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more) amino acid modifications. For example, a modified LGIC subunit described herein can have at least 75 percent sequence identity to SEQ ID N07 and can include a Q79G substitution and a A298G substitution. Additional examples of modifications that can confer the modified LGIC with selective binding of an ous LGIC ligand include modifications described elsewhere (see, e.g., US 8,435,762).

A modified LGIC subunit that selectively binds (e.g., enhanced g or increased potency) an exogenous LGIC ligand over an endogenous (e.g., a canonical) LGIC ligand can also be described as having enhanced potency for an exogenous ligand. In some cases, a d LGIC subunit described herein that ively binds an exogenous LGIC ligand can have at least 4 fold (e.g., at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, or at least 20 fold) enhanced potency for an exogenous ligand. In some cases, a modified LGIC subunit described herein that selectively binds an exogenous LGIC ligand can have 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) enhanced potency for an exogenous ligand. For example, a modified LGIC subunit described herein that selectively binds an exogenous LGIC ligand can have about 10 fold to about 100 fold enhanced y for an exogenous ligand. For example, a d LGIC subunit described herein that selectively binds an exogenous LGIC ligand can have about 10 fold to about 20 fold enhanced y for an exogenous ligand.

In some s, a modified LGIC subunit described herein can include at least one modified amino acid that confers the modified LGIC with reduced (e.g., minimized or eliminated) binding with an unmodified LGIC subunit. The binding with a modified LGIC subunit having the same modification can be selective over the binding with an unmodified LGIC subunit. An unmodified LGIC t can be a LGIC subunit lacking the ation that confers the modified LGIC with reduced binding with an unmodified LGIC subunit or an unmodified LGIC can be an endogenous LGIC subunit. The modification that confers the modified LGIC with reduced binding with an unmodified LGIC subunit can be a charge reversal modification. A modified LGIC subunit with d binding with an unmodified LGIC subunit can include any appropriate LBD (e.g., a 0L7 LBD). In some s, the modified LGIC t can include a 0L7 LBD set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO: 12, and the amino acid modification 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 an aspartic acid (e.g., R27D). For example, the ic acid at amino acid residue 41 of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO: 12 can be substituted with an arginine (e.g., E41R). In some cases, a modified LGIC subunit described herein can include a 0L7 LBD haVing a R27D substitution and a E41R.

In some aspects, a modified LGIC subunit bed herein can include at least one modified amino acid that confers the modified LGIC with reduced (e.g., minimized or eliminated) binding of an endogenous LGIC . The endogenous LGIC ligand can be ACh. A modified LGIC subunit with reduced binding of an endogenous LGIC ligand can include any appropriate IPD (e.g., a GlyR LBD). For example, the modified LGIC subunit can include a 0L7 LBD set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO: 12, and the amino acid ation can be a substitution at amino acid residue 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 can be substituted with a 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 tuted with an amino acid residue such as e (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 a glycine (e.g., Q139G) or a 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 a phenylalanine (e.g., Y210F). In some cases, the tyrosine at amino acid e 217 of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO: 12 can be substituted with a phenylalanine (e.g., Y217F). In some cases, the ate at amino acid residue 219 of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:11, or SEQ ID NO: 12 can be substituted with an alanine (e.g., D219A).

In some aspects, a modified LGIC subunit described herein can include at least one modified amino acid that confers the modified LGIC with increased ion conductance. In some cases, the modified LGIC subunit can include a 5HT3 IPD set forth in SEQ ID N03, and the amino acid modification can be a substitution at amino acid residue 425, 429, and/or 433. For example, a modified LGIC subunit described herein can e a 5HT3 IPD having a R425Q substitution, a R429D substitution, and a R433A substitution. In some cases, the modified LGIC subunit can include a 5HT3 IPD set forth in SEQ ID N04, and the amino acid modification can be a substitution at amino acid residue 420, 424, and/or 428.

For example, a d LGIC subunit described herein can e a 5HT3 IPD having a R42OQ substitution, a R424D substitution, and a R428A substitution.

In some cases, a modified LGIC described herein can include at least one ic 0L7-5HT3 LGIC subunit (SEQ ID NO:6) having a human 0L7 nAChR LBD (SEQ ID NO: 1) with a Q79G amino acid substitution and a Y115F amino acid substitution, and a murine 5HT3 IPD (SEQ ID N013).

In some cases, a modified LGIC described herein can include at least one chimeric T3 LGIC subunit (SEQ ID NO:6) having a human 0L7 nAChR LBD (SEQ ID NO: 1) with a Q79G amino acid substitution and a Q139G amino acid substitution, and a murine 5HT3 IPD (SEQ ID N013).

In some cases, a modified LGIC described herein can include at least one chimeric 0L7-GlyR LGIC subunit (SEQ ID NO:7) having a human 0L7 nAChR LBD (SEQ ID N02) with a Q79G amino acid substitution and a Y115F amino acid substitution, and a human GlyR IPD (SEQ ID N05) with a A298G amino acid substitution.

In some cases, a modified LGIC described herein can include at least one chimeric 0L7-GlyR LGIC subunit (SEQ ID NO:7) having a human 0L7 nAChR LBD (SEQ ID N02) with a Q79G amino acid substitution and a Q139G amino acid substitution, and a human GlyR IPD (SEQ ID N05) with a A298G amino acid substitution.

In some cases, a d LGIC described herein can e at least one chimeric 0L7-GlyR LGIC subunit (SEQ ID NO:7) having a human 0L7 nAChR LBD (SEQ ID N02) with a R27D amino acid substitution, a E41R amino acid substitution, a Q79G amino acid substitution, and a Y115F amino acid substitution, and a human GlyR IPD (SEQ ID N05) with a A298G amino acid substitution.

In some cases, a modified LGIC described herein can include at least one chimeric 0L7- GlyR LGIC subunit (SEQ ID NO:7) having a human 0L7 nAChR LBD (SEQ ID N02) with a substitution at amino acid residue 131 (e.g., L131Cg L131A, L131M, or L131N), and a human GlyR IPD (SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least one chimeric 0L7- GlyR LGIC t (SEQ ID NO:7) having a human 0L7 nAChR LBD (SEQ ID N02) with a substitution at amino acid residues 131 (e.g., L131Cg L131A, L131M, or L131N) and Y115 (e.g., Y115F), and a human GlyR IPD (SEQ ID NO:5).

In some cases, a modified LGIC bed herein can include at least one ic 0L7- GlyR LGIC subunit (SEQ ID NO:7) having a human 0L7 nAChR LBD (SEQ ID N02) with a substitution at amino acid es 131 (e.g., L131Cg L131A, L131M, or L131N) and 139 (e.g., , and a human GlyR IPD (SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least one chimeric 0L7- GlyR LGIC subunit (SEQ ID NO:7) having a human 0L7 nAChR LBD (SEQ ID N02) with a substitution at amino acid residues 131 (e.g., L131Cg L131A, L131M, or L131N) and 217 (e.g., Y217F), and a human GlyR IPD (SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least one chimeric 0L7- GlyR LGIC subunit (SEQ ID NO:7) having a human 0L7 nAChR LBD (SEQ ID N02) with a substitution at amino acid residues 131 (e.g., L131Cg L131A, L131M, or L131N), 139 (e.g., Q139L), and 217 (e.g., Y217F), and a human GlyR IPD (SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least one chimeric d7- 5HT3 LGIC subunit having a human 0L7 nAChR LBD (SEQ ID N02) with a substitution at amino acid residue 131 (e.g., L131Q L131A, L131M, or L131N), and a human 5HT3 IPD (SEQ ID NO:4).

In some cases, a modified LGIC described herein can include at least one chimeric 0L7- GlyR LGIC subunit (SEQ ID NO:7) having a human 0L7 nAChR LBD (SEQ ID N02) with a substitution at amino acid residue 175 (e.g, G175K), and a human GlyR IPD (SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least one ic d7- 5HT3 LGIC subunit having a human 0L7 nAChR LBD (SEQ ID N02) with a substitution at amino acid residue 131 (e.g., L131Q L131A, L131M, or L131N) and 139 (e.g., Q139L), and a human 5HT3 IPD (SEQ ID N04) with a R420Q tution, a R424D substitution, and a R428A substitution.

In some cases, a modified LGIC described herein can include at least one chimeric 0L7- 5HT3 LGIC subunit having a human 0L7 nAChR LBD (SEQ ID N02) with a tution at amino acid e 131 (e.g., L131Q L131A, L131M, or L131N) and 139 (e.g., Q139L) and 217 (e.g., Y217F), and a human 5HT3 IPD (SEQ ID N04) with a R420Q substitution, a R424D substitution, and a R428A substitution.

In some cases, a modified LGIC described herein can include at least one chimeric 0L7- GlyR LGIC subunit (SEQ ID NO:7) having a human 0L7 nAChR LBD (SEQ ID N02) with a substitution at amino acid residues 175 (e.g., G175K) and 115 (e.g., Y115F), and a human GlyR IPD (SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least one ic 0L7- GlyR LGIC subunit (SEQ ID NO:7) having a human 0L7 nAChR LBD (SEQ ID N02) with a substitution at amino acid residues 175 (e.g., G175K) and 115 (e.g., Y115F) and 79 (e.g., Q79G), and a human GlyR IPD (SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least one chimeric 0L7- GlyR LGIC subunit (SEQ ID NO:7) having a human 0L7 nAChR LBD (SEQ ID N02) with a substitution at amino acid residues 175 (e.g., G175K) and 77 (e.g., W77F) and 79 (e.g., Q79G), and a human GlyR IPD (SEQ ID NO:5).

In some cases, a modified LGIC described herein can e at least one chimeric 0L7- GlyR LGIC subunit (SEQ ID NO:7) having a human 0L7 nAChR LBD (SEQ ID N02) with a substitution at amino acid residue 216 (e.g., P2161), and a human GlyR IPD (SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least one chimeric 0L7- GlyR LGIC t (SEQ ID NO:7) having a human 0L7 nAChR LBD (SEQ ID N02) with a substitution at amino acid residues 216 (e.g., P2161) and 79 (e.g., Q79G), and a human GlyR IPD (SEQ ID NO:5).

In some cases, a modified LGIC described herein can include at least one chimeric 0t7-GlyR LGIC subunit (SEQ ID NO: 10) having a human 0L7 nAChR LBD (SEQ ID N02) with a substitution at amino acid residue 131 (e.g., L131A, L131Cg L131M, L131N, L131Q, L131V, or L131F), and a human GABAC IPD (SEQ ID N09).

In cases where a LBD and/or a IPD is a homolog, orthologue, or paralog of a sequence set forth herein (e.g., SEQ ID NOs: 1-5 and/or 9), it is understood that reference to a particular modified amino acid residue can shift to the corresponding amino acid in the homolog, orthologue, or paralog. For example, residues 425, 429, and 433 in a murine 5HT3 IPD set forth in SEQ ID NO:3 correspond to residues 420, 424, and 428 in a human 5HT3 IPD set forth in SEQ ID N04, and the R425Q, R429D, and R433A substitutions in a murine 5HT3 IPD correspond to R42OQ, R424D, and R428A substitutions in a human 5HT3 IPD.

Any method can be used to obtain a modified LGIC subunit described herein. In some cases, peptide synthesis methods can be used to make a modified LGIC subunit described herein. Examples of methods of e synthesis include, without limitation, liquid-phase peptide synthesis, and solid-phase peptide sis. In some cases, protein biosynthesis methods can be used to make a modified LGIC subunit described herein. es of methods of protein biosynthesis e, without limitation, transcription and/or translation of nucleic acids encoding a phosphorylation-mimicking peptide provided herein.

Similar modified LGIC subunits (e.g., modified subunits having essentially the same modifications and/or having essentially the same amino acid sequence) will self-assemble through interactions n the LBDs to form a d LGIC.

This document also provides nucleic acids encoding modified LGIC subunits described herein as well as constructs (e.g., plasmids, non-viral vectors, viral vectors (such as associated virus, a herpes simplex virus, or lentivirus vectors)) for sing nucleic acids encoding modified LGIC subunits described herein. Nucleic acids encoding modified LGIC subunits described herein can be operably linked to any appropriate promoter. A promoter can be a native (i.e., minimal) promoter or a composite promoter. A promoter can be a tous (i.e., constitutive) promoter or a regulated promoter (e.g., inducible, tissue c, cell-type specific (e.g., neuron c, muscle specific, glial specific), and neural subtype-specific). Examples of promoters that can be used to drive expression of nucleic acids encoding modified LGIC subunits bed herein include, without limitation, synapsin, CAMKII, CMV, CAQ enolase, TRPVl, POMC, NPY, AGRP, MCH, and Orexin promoters. In some cases, a nucleic acid encoding a d LGIC subunit described herein can be operably linked to a neuron specific promoter.

This document also provides cells (e.g., mammalian cells) having a modified LGIC described herein. ian cells having a modified LGIC described herein can be obtained by any appropriate method. In some cases, a pre-assembled modified LGIC can be provided to the cell. In some cases, a nucleic acid encoding a modified LGIC subunit described herein can be provided to the cell under conditions in which a modified LGIC subunit is translated and under conditions in which multiple (e.g., three, four, five, six, or more) modified LGIC ts can assemble into a d LGIC described herein.

LGIC Ligands This nt also provides LGIC ligands that can bind to and activate modified LGICs described herein. A LGIC ligand that can bind to and activate modified LGICs described herein can be exogenous or endogenous. A LGIC ligand that can bind to and activate modified LGICs described herein can be naturally occurring or tic. ALGIC ligand that can bind to and activate modified LGICs described herein can be canonical or non-canonical. A LGIC ligand that can bind to and te modified LGICs described herein can be an agonist or an antagonist. In some cases, an LGIC ligand is an exogenous LGIC t. Examples of LGIC ligands include, without limitation, ACh, nicotine, epibatatine, cytisine, RS56812, tropisetron, nortropisetron, PNU-282987, PHA-5436l3, compound 0353, compound 0354, compound 0436, compound 0676, compound 702, compound 723, nd 725, granisetron, ivermectin, mequitazine, promazine, varenicline, nd 765, nd 770, 3-(l,4-diazabicyclo[3.2.2]nonan yl)dibenzo[b,d]thiophene 5,5-dioxide, nd 773, and compound 774 (see, e.g., Figure 3B, Figure 5C, Figure 10A, and Figure 10B).

A LGIC ligand that can bind to and activate modified LGICs described herein can have selective binding (e.g., enhanced binding or increased potency) for a modified LGIC described herein. In some cases, a LGIC ligand that can bind to and activate modified LGICs described herein does not bind to and activate endogenous receptors. A LGIC ligand that selectively binds to and activates a modified LGIC (e.g., a modified LGIC having at least one amino acid modification that confers pharmacological selectivity to the modified LGIC) described herein over an unmodified LGIC ligand can also be described as having enhanced potency for a d LGIC. In some cases, a 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 for a modified LGIC. For example, a LGIC ligand that ively binds to and activates a modified LGIC can have 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) enhanced potency for a modified LGIC. 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 EC5O of less than 25 nM (e.g., less than 22 nM, less than 20 nM, less than 17 nM, less than 15 nM, less than 13 nM, less than 12 nM, less than 11 nM, or less than 10 nM) for a modified LGIC subunit described herein. For example, a LGIC ligand (e.g., etron) can have an EC5O of about 11 nM for a modified LGIC subunit described herein (e.g., d7Q79G-GlyRA298G). For example, a LGIC ligand (e.g., nortropisetron) can have an EC5O of about 13 nM for a modified LGIC subunit bed herein (e.g., 0L7Q79G’Y115F-GlyRA298G). In some cases, a LGIC ligand can have an EC5O of greater than 20 uM (e.g., greater than 22 nM, greater than 25 uM, greater than 35 uM, greater than 50, greater than 65 nM, greater than 80 uM, or r than 100 uM) for a modified LGIC subunit described herein. For e, a LGIC ligand (e.g., ACh) can have an EC5O of greater than 100 uM for a modified LGIC subunit bed herein (e.g. , 0L7Q79G,Y115F_G1yRA298G).

In some aspects, a LGIC ligand can be a synthetic ligand that can bind to and activate modified LGICs described herein can be a quinuclidine, a tropane, a 9- azabicyclo[3 .3. l]nonane, or a 2-phenyl-7,8,9, lO-tetrahydro-6H-6,lO-methanoazepino[4,5- g]quinoxaline.

A LGIC ligand that can be to and activate a modified LGIC described herein can have Formula I: (3 ML ém’V’Ex 7’ \gfi-‘F’X'E‘V—r ‘ \\ Xs‘m 5:! where X1 and X2 can independently be CH, CH2, 0, NH, or We, each n can independently be 0 or 1, Y can be 0 or S, A can be an aromatic tuent, and R can be H or pyridinymethylene. Examples of aromatic substituents include, without limitation, 4-chloro- e, lH-indole, 4-(trifluoromethyl) benzene, 4-chloro benzene, 2,5-dimethoxy benzene, 4-chloroaniline, aniline, fluoromethyl) pyridinyl, 6-(trifluoromethyl) nicotinic, and 4- chloro-benzene.

A LGIC ligand that can bind to and activate a modified LGIC described herein can be a quinuclidine. A quinuclidine can have the structure of Formula II: "4"" xx3 7 A Z. . if where X3 can be 0, NH, or CH2, Y can be 0 or S, A can be an ic substituent, and R can be H or pyridinylmethylene. Examples of aromatic substituents include without limitation, lH-indole, 4-(trifluoromethyl) benzene, 4-chloro benzene, 2,5-dimethoxy benzene, 4-(trifluoromethyl) benzene, 4-chloroaniline, aniline, 5-(trifluoromethyl) n yl, 6-(trifluoromethyl) nic, 3-chlorofluoro e, 4-chloro-benzene, and 1H- indole. Examples of quinuclidines include, without limitation, compounds PNU-282987, PHA-543613, 0456, 0434, 0436, 0354, 0353, 0295, 0296, and 0676 (see, e.g., Figure 5C, Table 3, and Table 6).

A LGIC ligand that can bind to and activate a modified LGIC described herein can be a tropane. A tropane can have the structure of Formula 111: where X2 can be NH or NMe, X3 can be 0, NH, or CH2, Y can be 0 or S, and A can be an ic substituent. Example of aromatic substituents include, without tion, lH- indole, 7-methoxy-lH-indole, 7-methyl-lH-indole, 5-chloro-lH-indole, and lH-indazole.

Examples of tropanes include, without limitation, tropisetron, pseudo-tropisetron, nortropisetron, compound 737, and compound 745 (see, e.g., Figure 5C, Table 3, and Table A LGIC ligand that can bind to and te a d LGIC described herein can be a 9-azabicyclo[3.3. l]nonane. A 9-azabicyclo[3.3. l]nonane can have the structure of Formula 2‘7"" ,‘x\\ K‘s)!" \ '3 {\‘yf , x A where Xl can be CH, X2 can be NH or NMe, X3 can be 0, NH, or CH, Y can be 0 or S, and A can be an aromatic substituent. An example of an aromatic substituent is, without limitation, 4-chloro-benzene. es of 9-azabicyclo[3.3.l]nonanes include, without limitation, compound 0536, compound 0749, compound 075l, compound 0760, and compound 0763 (see, e.g., Figure 5C, Table 3, and Table 6).

In some cases, a LGIC ligand can be an a 6,7,8,9-tetrahydro-6,10-methano-6H- pyrazino(2,3-h)benzazepine and can have a structure shown in Formula V: N A HN:in:\ N R where R = H or CH3, and where A = H or an aromatic substituent. Examples of 6,7,8,9- tetrahydro-6, hano-6H-pyrazino(2,3-h)benzazepines include, without limitation, cline, compound 0765, and compound 0770 (see, e.g., Figure 10A, Table 3, and Table In some cases, a LGIC ligand can be a l,4-diazabicyclo[3.2.2]nonane and can have a structure shown in Formula VI: WO 09832 where R = H, F, N02. Examples of l,4-diazabicyclo[3.2.2]nonanes include, without limitation, 3-(l,4-diazabicyclo[3.2.2]nonanyl)dibenzo[b,d]thiophene 5,5-dioxide, compound 0773, and compound 0774 (see, e.g., Figure lOB, Table 6, and Table 9).

Methods of Using This document also provides methods of using a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein. ALGIC ligand that can bind to and activate the modified LGIC can be used to activate a d LGIC with temporal and/or spatial l based on delivery of the .

In some aspects, a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein can be used to identify a ligand that selectively binds to a d LGIC described herein. For example, such screening methods can e providing one or more candidate ligands to a d LGIC bed herein, and detecting binding between the candidate ligand and the modified LGIC.

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

In some aspects, a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein can be used to treat a mammal having a channelopathy (e.g., a neural channelopathy or a muscle channelopathy). For example, a mammal having a channelopathy can be treated by administering a modified LGIC described herein, and then administering a LGIC ligand that can bind to and activate the modified LGIC. For example, a mammal having a channelopathy can be treated by administering a modified LGIC described herein (e.g., including at least one ic 0L7-GlyR LGIC subunit (SEQ ID NO:6) having a human 0L7 nAChR LBD (SEQ ID N02) with a R27D amino acid substitution, a E4lR amino acid substitution, a Q79G amino acid substitution, and a YllSF amino acid substitution, and a human GlyR IPD (SEQ ID N05) with a A298G amino acid substitution), and then administering tropisetron. For example, a mammal having a channelopathy can be treated by administering a modified LGIC described herein including a modified human 0L7 nAChR LBD (e.g., SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 1 l, or SEQ ID NO:12) with an Ll3l amino acid substitution (e.g., Ll3lG, Ll3lA, Ll3lM, or L13 1N) and, optionally, a Q79S amino acid substitution, a Ql39L amino acid substitution, and/or a Y217F amino acid substitution, and then administering varenicline, tropisetron, and/or compound 765.

Any type of mammal can be treated using a d LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein. For example, humans and other primates such as monkeys can be treated using a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein. In some cases, dogs, cats, horses, cows, pigs, sheep, rabbits, mice, and rats can be treated using a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein.

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

Once identified as having a channelopathy and/or a mammal at risk of developing a channelopathy, the mammal can be administered or instructed to self-administer a modified LGIC described , and then administered or instructed to self-administer a LGIC ligand that can bind to and activate the modified LGIC as bed herein. A modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein can be administered er or can be administered separately.

When treating a mammal having a channelopathy and/or a mammal at risk of developing a channelopathy using the materials and methods bed , the channelopathy can be any channelopathy. As used , a channelopathy can be any disease or disorder caused by aberrant ion channel function and/or aberrant ligand function, or which could be alleviated by modulated ion channel function and/or altered cellular ion fiux (e.g., calcium ion flux). A channelopathy can be congenital or acquired. Examples of lopathies include, without limitation, Bartter syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT), congenital nsulinism, cystic fibrosis, Dravet syndrome, episodic ataxia, erythromelalgia, generalized epilepsy (e.g., with febrile seizures), familial hemiplegic migraine, fibromyalgia, hyperkalemic periodic paralysis, hypokalemic ic paralysis, Lambert-Eaton myasthenic syndrome, long QT syndrome (e.g., Romano-Ward syndrome), short QT syndrome, malignant hyperthermia, mucolipidosis type IV, enia gravis, myotonia congenital, neuromyelitis optica, neuromyotonia, nonsyndromic deafness, paramyotonia congenital, retinitis pigmentosa, timothy syndrome, us, seizure, trigeminal neuralgia, and multiple sis.

Alternatively, or in addition, the materials and methods described herein can be used in other applications including, without limitation, pain treatment, cancer cell therapies, appetite control, spasticity treatment, muscle dystonia treatment, tremor treatment, and movement disorder treatment.

In some cases, a modified LGIC described herein and a LGIC ligand that can bind to and activate the d LGIC as described herein can be used to modulate the activity of a cell. The activity of the cell that is modulated using a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein can be any ar activity. Examples of cellular activities include, t limitation, active transport (e.g., ion transport), passive transport, excitation, inhibition, ion flux (e.g., calcium ion flux), and osis. The cellular activity can be increased or decreased. For example, a modified LGIC described herein and a LGIC ligand that can bind to and activate the d LGIC as described herein can be used to modulate (e.g., increase) ion transport across the membrane of a cell. For example, a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein can be used to modulate (e.g., increase) the excitability of a cell.

A d LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein can be used to te the activity of any type of cell in a mammal. The cell can be a neuron, a glial cell, a myocyte, an immune cell (e.g., neutrophils, eosinophils, ils, lymphocytes, and monocytes), an endocrine cell, or a stem cell (e.g., an nic stem cell). In some cases, the cell can be an excitable cell. The cell can be in vivo or ex vivo.

WO 09832 2017/041147 A modified LGIC described herein can be administered by any appropriate method.

A modified LGIC can be administered as modified LGIC subunits or as pre-assembled modified LGICs. A d LGIC can be administered as a nucleic acid encoding a modified LGIC. A modified LGIC can be administered as a nucleic acid encoding a d LGIC subunit described herein. For example, a nucleic acid can be red as naked nucleic acid or using any appropriate vector (e.g., a recombinant vector). Vectors can be a DNA based vector, an RNA based, or combination thereof. Vectors can express a nucleic acid in dividing cells or non-dividing cells. Examples of recombinant vectors include, without limitation, plasmids, viral vectors (e.g., retroviral vectors, adenoviral vectors, adeno-associated viral s, and herpes simplex vectors), cosmids, and artificial chromosomes (e.g., yeast artificial chromosomes or bacterial artificial chromosomes). In some cases, a nucleic acid encoding a modified LGIC subunit described herein can be sed by an adeno-associated viral vector.

A modified LGIC described herein can be detected (e.g., to confirm its presence in a cell) by any appropriate method. In some cases, an agent that ively binds a modified LGIC can be used to detect the modified LGIC. Examples of agents that can be used to bind to a modified LGIC described herein include, without limitation, antibodies, proteins (e.g., bungarotoxin), and small molecule ligands (e.g., PET ligands). An agent that selectively binds a modified LGIC can include a detectable label (e.g., fluorescent , radioactive labels, positron emitting , and enzymatic labels). Methods to detect LGIC expression in a cell can include fluorescence imaging, autoradiography, functional MRI, PET, and SPECT.

A modified LGIC described herein and a LGIC ligand that can bind to and activate the d LGIC as described herein can be administered to a mammal having a channelopathy and/or at risk of ping a channelopathy as a combination therapy with one or more additional agents/therapies used to treat a lopathy. For example, a combination therapy used to treat a mammal having a channelopathy as described herein can include administering a modified LGIC described herein and a LGIC ligand that can bind to and activate the d LGIC as described herein and treating with acetazolaminde, dichlorphenamide, mexilitine, glucose, m gluconate, L-DOPA, muscle stimulation, spinal stimulation, brain stimulation, and/or nerve stimulation.

In embodiments where a modified LGIC bed herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein are used in combination with additional agents/therapies used to treat a channelopathy, the one or more additional agents can be administered at the same time or independently. For example, a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein first, and the one or more additional agents administered second, or vice versa. In embodiments where a modified LGIC described herein and a LGIC ligand that can bind to and activate the d LGIC as described herein are used in combination with one or more additional therapies used to treat a channelopathy, the one or more additional therapies can be performed at the same time or ndently of the administration of a modified LGIC bed herein and a LGIC ligand that can bind to and activate the modified LGIC as described . For example, a modified LGIC described herein and a LGIC ligand that can bind to and activate the modified LGIC as described herein can be administered , , or after the one or more additional therapies are performed.

In some cases, a modified LGIC described herein and/or a LGIC ligand that can bind to and te the modified LGIC as described herein can be formulated into a pharmaceutically acceptable composition for administration to a mammal having a lopathy or at risk of developing a channelopathy. For example, a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC bed herein) and/or a LGIC ligand that can bind to and activate the modified LGIC as described herein can be formulated together with one or more pharmaceutically able carriers (additives) and/or diluents. A pharmaceutical composition can be formulated for administration in solid or liquid form including, without limitation, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules.

Pharmaceutically acceptable carriers, fillers, and vehicles that may be used in a pharmaceutical composition described herein e, without limitation, 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 ted vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

A pharmaceutical composition containing a modified LGIC described herein and/or a LGIC ligand that can bind to and activate the modified LGIC as described herein can be designed for oral, parenteral (including subcutaneous, intracranial, rterial, intramuscular, intravenous, intracoronary, intradermal, or topical), or inhaled stration.

When being administered orally, a pharmaceutical composition containing a therapeutically effective amount of a modified LGIC bed herein (e.g., a nucleic acid encoding a modified LGIC described herein) and/or a LGIC ligand that can bind to and activate the modified LGIC as described herein can be in the form of a pill, tablet, or capsule.

Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that can contain xidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and s and non-aqueous e suspensions which may e suspending agents and thickening agents.

Compositions for inhalation can be delivered using, for example, an r, a nebulizer, and/or a dry powder inhaler. The formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection ons and suspensions may be prepared from sterile powders, granules, and tablets.

A pharmaceutically acceptable composition ing a therapeutically ive amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and/or a LGIC ligand that can bind to and activate the modified LGIC as bed herein can be administered locally or systemically. In some cases, a composition containing a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a d LGIC described herein) and/or a LGIC ligand that can bind to and activate the modified LGIC as described herein can be administered systemically by venous or oral administration to, or inhalation by a mammal (e.g., a human). In some cases, a composition containing a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and/or a LGIC ligand that can bind to and activate the modified LGIC as described herein can be administered locally by aneous, aneous, intramuscular, intracranial, or open surgical administration (e.g., injection) to a target tissue of a mammal (e.g., a human).

Effective doses can vary depending on the severity of the channelopathy, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other eutic treatments such as use of other agents, and the nt of the treating physician.

The frequency of stration can be any frequency that es symptoms of a channelopathy without producing significant toxicity to the mammal. 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 be variable during the duration of treatment. A course of treatment with a composition containing a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and/or a LGIC ligand that can bind to and te the modified LGIC as described herein can include rest periods. For example, a composition containing a therapeutically effective amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and/or a LGIC ligand that can bind to and activate the 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 influence the actual frequency of administration used for a particular application. For example, the effective amount, on of treatment, use of multiple treatment agents, route of administration, and ty of the channelopathy may require an increase or decrease in administration ncy.

An effective duration for administering a composition containing a therapeutically ive amount of a modified LGIC described herein (e.g., a nucleic acid encoding a modified LGIC described herein) and/or a LGIC ligand that can bind to and activate the modified LGIC as described herein can be any duration that improves symptoms of a channelopathy without producing significant toxicity to the mammal. For example, the effective duration can vary from several days to several weeks, months, or years. In some cases, the effective on for the ent of a lopathy can range in duration from about one month to about 10 years. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the ncy of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the channelopathy being treated.

In certain ces, a course of treatment and the symptoms of the mammal being treated for a channelopathy can be monitored. Any appropriate method can be used to monitor the symptoms of a channelopathy.

The invention will be further described in the following examples, which do not limit the scope of the invention bed in the claims.

EXAMPLES Example I .' Potency-enhancing ligand binding domain mutations A screen was performed with a panel of 41 d7-5HT3 chimeric channels having mutant LBDs against a panel of 51 clinically used drugs with chemical similarity to nicotinic receptor agonists. Mutations were at residues highlighted in Figure l. The screen revealed mutations at Gln79 in the 0L7 nAChR LBD that enhanced potency for the known nAChR agonist tropisetron (Figure 2). These mutations (Q79A, Q79G, Q79S) reduce the size of the amino acid side chain. Some mutant ion l-ligand combinations gave up to 12-fold improvement in y (Table 1, Figure 3). Canonical 0L7 nAChR agonists, ACh, ne, epibatidine, and the anti-smoking drug varenicline were not significantly affected by Q79A, Q79G, or Q79S mutations. However, a subset of 0L7 nAChR agonists showed enhanced potency with some of the mutations. Cytisine, RSS6812, tropisetron, nortropisetron, and PNU-282987 showed significantly ed potency for 0L7Q79G-5HT3. Additionally, nortropisetron and PNU-282987 showed a significantly enhanced potency for d7Q79A-5HT3 and d7Q79S-5HT3, respectively. In general, agonists based on a lidine or tropane pharmacophore with a linked ic structure that interacts with the complementary binding face of the ligand binding domain showed improved potency with Gln79 substitution with the smaller amino acid residues Ala, Gly, or Ser. For most agonists, d7Q79G-5HT3 was the most preferred mutant chimeric ion channel.

Table l. y of nAChR ts against chimeric cation channels mutated at Gln79 in HEK cells. Mean ECSO, SEM in parentheses (uM). 3 u7Q79A-5HT3 u7Q79G-5HT3 a7Q79S—5HT3 WO 09832 choline 7.0 (0.8) 6.2(1.4) ne 3.9 (0.4) 2.1 (0.4) Epibatidine 0.053 (0.006) 0.044 (0.006) Varenicline 0.92 (0.16) 0.47 (0.07) Cytisine 8.2 (0.3) 4.4(10) RS56812 10(18) 5.7 (0.8) Tropisetron 0.24 (0.03) 0.11 (0.02) Nortropisetron 0.061 (0.021) 0.019 (0.007) PNU-282987 0.22 (0.03) 0.023 (0.004) These mutated LBDs were used to generate 017-GlyR chimeric channels having ed potency for most of these ligands up to 6-fold (Figure 4A). Like mutations of 017- 5HT3, these mutations at G1n79 did not cantly affect potency of ACh, ne, epibatidine, varenicline, or cytisine. However, tropisetron, nortropisetron, and RSS6812 showed significantly enhanced potency for G-GlyR. Similar to LED mutations for 017- 5HT3, nortropisetron had significantly enhanced potency for 017Q79A-GlyR, and PNU-282987 showed significantly enhanced potency for 017Q79S-GlyR. For most agonists, G-GlyR was the most preferred mutant chimeric ion channel.

Another relationship that was observed in the small molecule screen was that mutations at Trp77 conferred agonist activity for the drug granisetron at the 017W77F-5HT3 (ECSO: 1.2 uM),017W77Y-5HT3 (ECSO: 1.1 0M), and 0L7W77F-GlyR (ECSO: 0.66 11M) ors. Granisetron is a 5HT3 receptor antagonist granisetron, which does not activate 017- 5HT3 or 0t7-GlyR.

These results show that mutation of Q79 (to A, G, or S) in the 017 nAChR LBD enhanced binding of known LGIC s to modified LGICs.

Example 2.‘ Potency enhancing ion pore domain mutations 017-GlyR channels having IPD ons previously established in full length glycine receptor channels (T258S and A288G, GlyR numbering, equivalent to T268S and A298G for 017-GlyR numbering) were examined for enhanced potency for the allosteric agonist ivermectin. Channels having yRT2688 were found to have substantial ligand-free open probability, which rendered them unsuitable for ligand-controlled manipulations of cells.

Mutations at 017-GlyRA298G, which were ive for enhancing ivermectin potency at the full length glycine receptor, led to modest change in open probability in the absence of the ligand, thus this channel was examined for activity against a panel of known agonists. For cal agonists ACh, nicotine, and epibatidine, as well as for varenicline and tropisetron, the agonist potency was not significantly enhanced in 0L7-GlyRA298G. A subset of 0L7 nAChR agonists did show up to a modest 4-fold increase in potency: RSS6812, cytisine, PNU- 282987, and nortropisetron were significantly more potent. Therefore, the effect of the IPD A298G mutation improved ligand potency, but depended on ligand ure and was not as effective as mutations in the LBD.

The Q79G mutation in the LBD and the A298G IPD mutation for 0L7-GlyR was examined (Table 2). The double mutant chimeric channel, 0L7Q79G-GlyRA298G, led to synergistic enhancement of potency showing up to 18-fold ement of potency relative to 0L7-GlyR to a7 nAChR agonists. The enhancement from this double mutant channel was greater than that from the individual mutations for agonists RSS6812, tropisetron, nortropisetron, and PNU-282987. Further underscoring the unexpected structural sensitivity of this combination of mutations, multiple agonists, such as ACh, nicotine, epibatidine, varenicline, and cytisine were not significantly changed between 0L7-GlyR and 0L7Q79G- GlyRA298G. Therefore, combination of the LBD on Q79G with the IPD mutation A298G led to a synergistic effect where potency for some but not all nic agonists was y increased by -fold.

Table 2. Potency of nAChR agonists against mutated chimeric chloride channels. Mean ECSO and SEM in parentheses (uM) for agonist activity in HEK cells expressing ic channels.

Agonist (x7 GlyR u7Q79A-GlyR u7Q79G-GlyR u7Q795-GlyR ()t7-GlyRAmG - GlyRA298G Acetylchoune4————_8 (05> Nicotine 2————_2 Epibandme o————_032 Varemcnne o————_33 mm mm» Rsm o————_61 etron O————_011 (0.002) Nomopisetron0.002(0.001) PNU—2829870.007(0.001) These results show that mutation of Q79 (to A, G, or S) in the 0L7 nAChR LED and/or mutation of A298 (to G) in the GlyR IPD further ed ive binding of known LGIC ligands to modified LGICs.

Example 3 .' Molecules exhibiting enhancedpotency Based on the ure activity relationship of known agonists that showed enhanced potency with G-GlyRA298G, a variety of synthetic molecules comprised of either lidine, tropane, or 9-azabicyclo[3.3.l]nonane pharmacophores with one or more aromatic side chain substituents were tested. In addition, the known 0L7 nAChR agonist FHA-543613 (Walker et al 2006, Wishka et al 2006) was also tested and showed exceptional potency for 0L7Q79G-GlyRA298G. These molecules generally showed enhanced potency 10-fold to 100-fold (Table 3), indicating that, for these pharmacophores, a range of structural es were compatible with improved potency for d7Q79G-GlyRA298G.

These results show that modified LGICs can be activated by synthetic quinuclidine- containing and tropane-containing LGIC ligands. wamfikwm—Udflona 9338M 3352" so N80 go 0 0 N80 Q: 3 Enema 933% II N80 h. 2 ooMA M80 II MEEmIBS 9338M Mull 8M :II mumofiqoém A 252.20%- EEE: 202% QESEBOMMEEY? oMMONMMon : @3686- see oMMmNMMon oMMoNMMon AMEBEBOEEV- I'm: 2mm I mm m -MMMUFE 08:88 :32 335% MJU was; 222:2? Enema :6 oEoEEo Eu 2&2 :6 Hmfimwm EEE IIIIII EOIIIIIIIII MHIIIIIIIIIIIIIII III!!!-xMMMM EIEéIIIIII MOOIIIIIIIII NOOOIIIIIIIII qusomaoo 222:2? EEE wIIIIIIHOsagéoaiIIIIII O///////// o o o o o N5 £0 £0 WI£0 53:5 oEoEEo z U .m wfimmomeo ungaacv 50%- mjomwm 28; DE 0% Eob bemEoEoZ In \0 Ca\ Ca\ E Acetylcholine responsiveness was considerably reduced to more than 100 uM in some cases with additional LBD mutations Y115F and Q139G that that only modestly d the potency of some ts for 0L7Q79G’Y115F-5HT3, 0L7Q79G’Q139G-5HT3, 0L7Q79G’Q139G-GlyRA298, 0L7Q79G’Y115F-GlyRA298G. For example, 0t7Q79CLY115F-GlyRAZQgG has an EC50 of 13 nM for nortropisetron and >100 uM for ACh (Table 4).

Table 4. Potency of nAChR agonists against mutated chimeric chloride channels with low acetylcholine responsiveness. Mean EC50 and SEM in parentheses (uM) for activity in HEK cells expressing chimeric channels. u7Q79G,Y115F_ u7Q79G,Q139G_ u7Q79G,Y115F_ u7Q79G,Q139G_ u7R27D,E41R,Q79G,Y115F_ Acetylchonne Nicotine 0.086 Troplsetron. 0.10(0.12) 0.31(0.06) 0.26(0.04) 0.035 (0.021) (0 043) 0.028 0.047 0.013 0.031 Nomepisetron (0.005) (0.013) (0.001) (0.006) 0.003 (0.001) PNU-282987 0.35(0.07) 0.16(0.04) 4) .04) 0.066(0.010) These results show that Y115F and/or Q139G mutations in the 0L7 nAChR LBD reduced binding of the endogenous LGIC ligand Ach to the modified LGIC.

Example 5.' Mutations that reduce associations with nous or subunits Assembly of 0L7 nAChRs is based on ations of five homomeric ts through ctions between the LBDs (Celie et al 2004 Neuron 41: 907-14). To minimize undesired associations with endogenous 0L7 nAChR subunits and/or unwanted ations of chimeric channels, potential inter-subunit salt bridges were fied by examining the crystal structure of the acetylcholine binding protein and identifying nearby inter-subunit residues with opposite charge that also have homologous ionic amino acids in the 0L7 nAChR or LBD. Charge reversal mutations (switching the acidic member of a potential salt bridge to a basic residue and its basic partner to an acidic residue) were designed to t inter-subunit interactions with unmodified subunits but preserve interactions between the subunits with charge al mutations (Figure 6A). Chimeric LGIC subunits having charge reversal mutations were able to assemble selectively with each other without interacting with unmodified channels, e.g. nous a7 nAChR. The double mutation of R27D,E41R in the (17 nAChR LBD resulted in onal channels (Figure 6B). Co-eXpression of these charge reversal channels with d7-5HT3 channels having an unmodified sequence showed that the charge reversal subunits did not co-immunoprecipitate with unmodified channels e 6C). Combination with potency enhancing mutations and acetylcholine blocking mutations to give the chimeric l 0L7R27D’E4IR’Q79G’Y115F-GlyRA298G revealed that some agonists retained high y for their cognate agonist (Table 4, right ).

These results show that R27D and E4lR mutations in (17 nAChR LBD reduced association of the modified LGIC ts with other modified and/or endogenous LGIC subunits.

Example 6: LED ons that increase ligandpotency Mutations in Gly175 and Pro216 of the 0L7 nAChR LED in 0L7-GlyR chimeric channels were tested. Mutation of Gly175 to Lys (0L7G175K-GlyR) showed increased potency for ACh (5- fold) (Table 5). For 0L7G175K-GlyR, it was also found that nicotine potency was enhanced 10- fold relative to the unmodified 0L7-GlyR chimeric channel (Table 5). Mutation of Pro216 to Ile (0L7P216I-GlyR) did not substantially alter ACh potency (Table 5). However, d7P216I-GlyR showed increased nicotine potency by >4-fold relative to fied 0L7-GlyR (Table 5).

These potency enhancing mutations in 0L7G175K-GlyR and 0L7P216I-GlyR also affected potency of several other 0L7-GlyR agonists up to 30-fold (Table 5). For a7G175K-GlyR, greater than 10- fold potency enhancement over 0L7-GlyR was seen for the clinically used drugs tropisetron, varenicline, cytisine, etron, and epibatidine. For 0t7P216I-GlyR, potency enhancement was approximately 3-fold (Table 5). 0000000 005 00000 00000 mm Wm 003.0 00.0 wN.m mod mofio Sod 0N00 00A '13 0000000 El. —1 00 0R. Hm. b2. who. 000. mm '13 m 0 /\ 00 o 0 0 0 000 0000000 00.N:aA bNd 00 000. woo. moo. 000. 00 0 0 0 0 0000000 0000A 00A Ag 0 0A 0:0. '13 0020000000 0000000 A0300 AN.0000 9:00 —1 00 A00. R NN. v 0 o AN00. NS. A000.

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To develop ls with reduced ACh responsiveness but high potency for other agonists, 5K-GlyR was combined with additional mutations that increase the potency of specific agonists. Combination with W77F reduced ACh potency, and 0L7W77F’G175K-GlyR showed increased potency over 0L7-GlyR for etron, pisetron, and tropisetron but not for PNU282-987, varenicline, cytisine, or FHA-543613 (Table 5). Combination of G175K with Q79G reduced ACh potency, and d7Q79G’G175K-GlyR showed increased potency for nortropisetron, FHA-543613, and tropisetron (Table 5). However, this potency enhancement was not observed for other agonists, such as PNU282-987, or varenicline. 5K’Q139L-GlyR reduced ACh potency and increased potency for nortropisetron and etron (Table 5).

Further reductions in ACh potency were achieved while maintaining high potency for with synthetic ts, including those based on tropane and quinuclidine core structures, by incorporating mutations at W77F, Q79G, Ll4lF, Yl 15F, Gl75K, and Y2lOF in various combinations. 0L7Q79G’YHSF’Gl75K-GlyR reduced ACh responsiveness while maintaining potent responses to tropisetron (Table 5). These mutations also enhanced responsiveness to other tropane and quinuclidine core structures relative to 0L7Y115F’G175K-GlyR as well as relative to d7-5HT3 (representative of endogenous 0L7 nAChR activity), especially quinuclidine thioureas 702 and 703 as well as tropane ester 723, 725, 726, 736, 737, 738, and 745 (Table 6). d7Q79G’Y115F’Gl75K-GlyR also showed high sensitivity to ivermectin (Table 5). d7W77F’Q79G’GI75K-GlyR reduced ACh responsiveness while ining high y responses to tropisetron, and nortropisetron (Table 5). d7W77F’Q79G’GI75K-GlyR also showed enhanced potency for additional tropane-based core structures, such as compounds 723 and 725, as well as the clinically used drugs mequitazine and promazine (Table 6). d7W77F’G175K’Y210F-GlyR reduced ACh responsiveness but markedly improved potency to granisetron (Table 5). 0L7L141F’Y115F’Gl75K-GlyR reduced ACh responsiveness while conferring sensitivity to granisetron (Table 5). d7Q79G’QI39L’GI75K-GlyR reduced ACh responsiveness but showed potent ses to nortropisetron (Table 5).

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Combination with non-association mutations to form 0L7RZ7D’E4IR’Q79G’Y1"EGl75K further improved the potency for 702, 723, 725, and 726, with low ACh responsiveness (Table 6).

Table 7. Agonist potency enhancement by G175K and A298G mutations at d7GlyR chimeric channels as well as W298A at 0L7GABAC (also referred to as GABAA-p) channels. nd 0L7GlyR 0L7GlyR a7GlyR a7GlyR a7GlyR a7GABAc a7GlyR G175K Y115F Acetylchonnemm Epibandme Varemcnne PNU—282987 Gramsetron nd = not determined; parentheses: SEM onal amino acid substitutions at Gly175 of the 0L7 nAChR LED in 5F -GlyR chimeric channels are also enhanced agonist potency. Potency for etron at 0L7Y115F — GlyR chimeric channels was enhanced with additional mutations, which include G175A (7.1- fold), G175F (2-fold), G175H (23-fold), G175K (56-fold), G175M (26-fold), G175R (5.8- fold), G175S (93-fold), G175V (16.7-fold).

Table 8. t potency enhancement by G175 mutations at d7GlyR Y115F chimeric channels.

Compound a7GlyR 0L7GlyR R 0L7GlyR 0L7GlyR 0L7GlyR 0L7GlyR 0L7GlyR R Y115F Y115F Y115F Y115F Y115F Y115F Y115F Y115F G175K G175A G175F G175H G175M G175R G175S G175V Varenicline III-I...-0.62 5.0 (1.7) 5.9 13.6 12.7 14.1 7.6 9.7 4.6 Tropisetron III-I...-0.15 0.027 0.021 0.074 0.064 0.057 0.024 0.016 0.009 PHA-543613 III-I...-0.03 0.02 0.027 0.173 0.12 0.25 0.11 0.12 0.037 nd = not ined; parentheses: SEM Mutations for Leu131 to smaller amino acids were found to reduce the potency of canonical agonists ACh and nicotine, while markedly increasing potency of varenicline, etron and several other agonists. 0L7L131A-GlyR and 0L7L131G-GlyR had reduced ACh responsiveness (6-fold) and enhanced potency for varenicline d and 17-fold, respectively) and tropisetron (25-fold and 36-fold, respectively) (Table 9). d7L131G-5HT3 HC had reduced ACh responsiveness (5-fold) and enhanced y for varenicline (16-fold) and tropisetron (23-fold) (Figure 9A and Table 9). 0L7L131G’Q139L-GlyR and 0L7L131G’Y217F-GlyR showed similar potency enhancement over 0L7-GlyR for varenicline (21-fold) but also reduced ACh sensitivity (-1 l-fold and -l3-fold, respectively). d7Q79S’L131G-GlyR further improved y over 0L7-GlyR for varenicline (89-fold) and tropisetron (15-fold). d7L131G’Q139L’Y217F-GlyR showed the greatest improvement in y over 0L7-GlyR for varenicline (3 87-fold) and also showed reduced ACh potency (13-fold) (Figure 9B and Table 9). d7L131G’Q139L’Y217F-GlyR also showed extremely high potency for compound 770 (0.001 uM), compound 773 (0.00034 uM), and compound 774 (0.00013 uM) (Figure 10). 0L7Q798’L131G’Q139L-GlyR also improved potency over 0L7-GlyR for varenicline (31-fold) and etron (3 -fold) but reduced ACh y (9-fold) (Figure 9B and Table 9). d7L131MGlyR , d7L131Q-GlyR, and 1V-GlyR reduced ACh potency but enhanced potency to tropisetron, nortropisetron, PHA-5436l3, and granisetron (Table 9). 0L7L131F-GlyR was found to ntially reduced ACh potency but did not improve potency for other agonists (Table 8). 0L7L131G-GABAC substantially reduced ACh potency but did not improve potency for other agonists (Table 9). d7L131G’Q139L’Y217F-5HT3 HC (Table 9) improved varenicline potency by 13 l-fold over a7-5HT3 (Table l). d7L131G’Q139L’Y217F-5HT3 HC also showed high potency for compound 770 (0.007 uM), nd 773 (0.002 uM), and compound 774 (0.004 uM) (Table 8). "a E E EEE O5: v—4 0 "CS "CS "CS "CS v—4 N Q Q Q o Boo Nooo vooo EEE O5: mm m: mooo v—4 v—4 mmoo oooo mooo Soo U: U: U: U: EOE >5: E2 goo? A goo goo Nooo E moo.o :oo N Im.ooo v—4 V U: U: IU: IU: V o EOE 9:: mm m: Eoo 0 v mo /\ goo wvoo mooo Boo U: A oooo Nooo EOE ES: Em Io: Io: o: U: .

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A low concentration of tropisetron (30 nM or 100 nM) was administered to mouse cortical neurons. Neuron activity was silenced by application of low concentration of agonist (Figure 7 and Figure 8C).

DNA plasmids containing nucleic acids encoding a 0L7Ll3 lG,Ql39L,Y217F-GlyR chimeric LGICs were transfected into mouse cortical neurons. Low concentration of varenicline (10 nM) was administered to mouse cortical s. Neuron activity was silenced by application of low concentration of agonist (Figure 9C).

These results show that modified LGIC activity can be controlled in neurons using low concentration of the LGIC ligands tropisetron and cline.

Example 8: ic LGICS in therapy Chemogenetic tools offer an tive strategy for combined drug and gene therapy.

This is e cellular function can be modulated in a consistent manner across different cell types in various tions using the same ion channels and ligands by use of an exogenously delivered ion channel that is selectively engaged by administration of a drug.

Identification of ion channels that are gated by well tolerated, clinically used drugs are especially attractive for potentially extending chemogenetics to human therapeutic use.

For the drug tropisetron, we have found that it activates 0L7Q79G-GlyRAZ98G with an ECSO of 11 nM, which is similar to the reported ICSO of 10 nM tropisetron for its therapeutic target, the 5HT3 or (Combrink et al 2009 Pharmacological reports: PR 61: 785-97).

OTHER EMBODIMENTS It is to be understood that while the sure has been described in conjunction with the detailed description thereof, 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 s, advantages, and modifications are within the scope of the following claims.

Claims (45)

WHAT Is CLAIMED Is:

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

2. The modified LGIC of claim 1, n the modified LGIC is a ic LGIC comprising a LBD from a first LGIC and an IPD from a second LGIC.

3. The modified LGIC of claim 1, wherein the LBD is an alpha7 nicotinic acetylcholine receptor (017-nAChR) LBD.

4. The modified LGIC of claim 3, wherein the amino acid modification comprises an amino acid tution 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 ofthe 017-nAChR LBD.

5. The d LGIC of claim 4, wherein the amino acid tution is at residue 77 of the 017-nAChR LBD, and wherein the amino acid substitution is selected from the group ting of W77F and W77Y.

6. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 79 of the 017-nAChR LBD, and wherein the amino acid substitution is selected from the group consisting of Q79A, Q79Cg and Q79S.

7. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 115 of the 017-nAChR LBD, and wherein the amino acid substitution is a Y115F tution.

8. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 131 of the 017-nAChR LBD, and wherein the amino acid substitution is selected from the group consisting ofL131A, L131Q L131M, and L131N.

9. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 139 of the d7-nAChR LBD, and n the amino acid substitution is selected from the group consisting on139G and Q139L.

10. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 175 of the d7-nAChR LBD, and wherein the amino acid substitution is ed from the group consisting of G175A, G175F, G175H, G175K, G175M, G175R, G17SS, and G175V.

11. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 210 of the d7-nAChR LBD, and wherein the amino acid substitution is a Y210F substitution.

12. The modified LGIC of claim 4, wherein the amino acid substitution is at residue 216 of the d7-nAChR LBD, and wherein the amino acid substitution is a P2161 substitution.

13. The modified LGIC of claim 4, n the amino acid substitution is at residue 217 of the d7-nAChR LBD, and wherein the amino acid substitution is a Y217F substitution.

14. The modified LGIC of claim 4, wherein the amino acid tution is at residue 219 of the d7-nAChR LBD, and n the amino acid substitution is a D219A substitution.

15. The modified LGIC of claim 4, wherein the 0L7-11AChR LBD comprises a L131G amino acid tution, a Q139L amino acid substitution, and a Y217F amino acid substitution.

16. The modified LGIC of claim 4, wherein the 0L7-11AChR LBD comprises a L131M amino acid substitution and a Y115F amino acid substitution.

17. The modified LGIC of claim 4, wherein the 0L7-11AChR LBD comprises a W77F amino acid substitution, a Q79G amino acid substitution, and a G175K amino acid substitution.

18. The modified LGIC of claim 4, wherein the 0L7-nAChR LBD comprises a Q79G amino acid substitution, a Y115F amino acid substitution, and a G175K amino acid substitution.

19. The modified LGIC of claim 4, n the 0L7-nAChR LBD comprises a Y115F amino acid substitution and a G175K amino acid substitution.

20. The modified LGIC of claim 4, wherein the 0L7-nAChR LBD ses a Q79G amino acid substitution and a 2161 amino acid substitution.

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 gamma-aminobutyric acid (GABA) receptor IPD, and an alpha7 nicotinic acetylcholine receptor (0L7-nAChR) IPD.

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

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

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

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

26. The d LGIC of claim 25, wherein the synthetic exogenous LGIC ligand is a tropane, and n the tropane is selected from the group consisting of tropisetron, pseudo- tropisetron, nortropisetron, compound 723, compound 725, compound 737, and compound

27. The modified LGIC of claim 25, wherein the tic exogenous LGIC ligand is a quinuclidine, wherein the quinuclidine is selected from the group consisting of PNU-282987, PHA-5436l3, compound 0456, compound 0434, compound 0436, compound 0354, compound 0353, compound 0295, compound 0296, compound 0536, compound 0676, and compound 702.

28. The modified LGIC of claim 25, wherein the synthetic exogenous LGIC ligand is a 9- azabicyclo[3.3.l]nonane, and wherein the 9-azabicyclo[3.3. l]nonane is compound 536.

29. The modified LGIC of claim 25, wherein the synthetic ous LGIC ligand is a 6,7,8,9-tetrahydro-6,l0-methano-6H-pyrazino(2,3-h)benzazepine, and wherein the 6,7,8,9- tetrahydro-6, l0-methano-6H-pyrazino(2,3-h)benzazepine is selected from the group consisting of varenicline, compound 765, and compound 770.

30. The modified LGIC of claim 25, wherein the synthetic exogenous LGIC ligand is a 1,4- diazabicyclo[3.2.2]nonane, and n the a l,4-diazabicyclo[3.2.2]nonane is selected from the group consisting of 3-(l,4-diazabicyclo[3.2.2]nonanyl)dibenzo[b,d]thiophene 5,5- e, compound 773, and compound 774.

31. 3 l. The modified LGIC of claim 1, wherein the LBD is a 0L7-nAChR LBD, and wherein the d7-nAChR LBD further comprises at least one modified amino acid that confers selective binding to r d7-nAChR LBD haVing the at least one modified amino acid over binding to an unmodified LGIC.

32. The d LGIC of claim 3 1, wherein the fied LGIC is an endogenous LGIC.

33. The modified LGIC of claim 32, wherein the endogenous LGIC is an endogenous d7- nAChR.

34. The modified LGIC of claim 3 1, n the at least one modified amino acid that confers selective g comprises an amino acid substitution at an amino acid residue at e 27 and/or residue 41 of the d7-nAChR LBD.

35. The modified LGIC of claim 34, wherein the at least one modified amino acid comprises a R27D substitution and/or a E4lR substitution.

36. The modified LGIC of claim 1, wherein the IPD is a murine 5HT3 IPD, and wherein the murine 5HT3 IPD r comprises at least one modified amino acid that confers increased ion conductance to the modified LGIC.

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

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

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 ion conductance to the modified LGIC.

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

41. The modified LGIC of claim 40, wherein at least one modified amino acid comprises a R42OQ substitution, a R424D substitution, and/or a R428A substitution.

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

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

44. The modified LGIC of claim 43, wherein the modified LGIC has an ECSO of greater than 20 uM for Ach.

45. A ligand having increased potency for a modified ligand gated ion channel (LGIC), wherein the ligand comprises Formula I: C39") “‘"l‘lxxcx \M X1 5‘ {m EN\