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Cation conducting gabaa receptors and their use

a gabaa receptor and cation conducting technology, applied in the field of cation conducting gabaa receptors and mutated gabaa receptor subunits, can solve the problems of inability to maintain a sufficient chloride gradient across the membrane of most cells capable of functionally expressing gabaa receptors, incompatible with conventional hts methods, and inability to affect modulator sites, etc., to achieve high throughput drug screening

Inactive Publication Date: 2008-02-07
JENSEN MARIANNE LERBECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The GABAA receptor, however, is a chloride channel, rendering it incompatible with conventional HTS methods.
Moreover, most cells capable of functionally expressing GABAA receptors do not maintain a sufficient chloride gradient across their membranes.
This receptor, however, does not contain modulator sites affected by drugs, and is not suited for high throughput drug screening.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Mutagenesis and Expression of Mutated Receptors

[0069] To investigate the influence of the GABAA receptor subunits in defining the ion selectivity of the receptor, a mutant GABAA receptor subunit 3 was designed by site directed mutagenesis.

[0070] The GABAA receptor subunits were cloned by RT-PCR from human brain mRNA (Clontech). The GABAA receptor α subunit was sub-cloned into the pNS1z vector, the GABAA receptor β subunit was sub-cloned into the pNS1n vector, and the GABAA receptor γ subunit was sub-cloned into the pZeoSV vector (Invitrogen). pNS1n and pNS1z were derived from pcDNA3-Neo (Invitrogen) or pcDNA3-Zeo (Invitrogen), respectively.

[0071] A hindlll site was introduced in the loop between the Ml and M2 domains and a bsu36l site was introduced in the loop between the M2 and M3 domains in all receptor subunits. hindill and bsu36l sites at other positions within the vectors or cDNAs were eliminated.

[0072] A set of chimeric GABAAR subunits was constructed in which the M2 dom...

example 2

Expression of Mutated Receptors

[0080] All constructs were expressed in CHO-K1 cells (ATCC No. CCL61).

[0081] CHO-K1 cells were maintained in DMEM with 10 mM HEPES and 2 mM glutamax supplemented with 10% fetal bovine serum and 2 mM L-proline (Life Technologies). The cells were cultured at 37° C. in a humidified atmosphere of 5% CO2 and 95% air and passaged twice a week.

[0082] CHO-K1 cells were co-transfected with the plasmids described above and a plasmid encoding enhanced green fluorescent protein using the lipofectamine PLUS-kit (Life Technologies) according to manufacturers protocol.

[0083] Binding experiments and electrophysiological measurements were performed 24-48 hours after transfection.

example 3

Binding Assays

[0084] Binding studies were performed using standard methods. Membranes were prepared from CHO-K1 cells expressing recombinant GABAAR subunits. The cells were washed in PBS (Life Technologies), trypsinized, washed twice in tris-citrate buffer (50 mM, pH 7.1) and centrifuged for 10 minutes at 5,000 g. [3H]-Muscimol binding

[0085] Membranes were resuspended in membrane wash buffer [20 mM KH2PO4 / K2HPO4, pH 7.5, 50 mM KCl, 0.025% (w / v) NaN3, and various protease inhibitors (1 mM EDTA, 2 mM benzamidine chloride, 0.1 mM benzethonium chloride, 50 U / ml bacitracin, 0.3 mM phenylmethylsulfonyl fluoride, 10 mg / l ovomucoid trypsin inhibitor, 10 mg / l soybean trypsin inhibitor)] and centrifuged for 30 minutes at 177,000 g and 4° C. The pellet was resuspended in binding assay buffer (20 mM KH2PO4 / K2HPO4, pH 7.5 and 100 mM KCI) to a protein concentration of 1 mg / ml and homogenized just before use. Binding was performed with 1, 3, 10, 30, 100 or 300 nM of [3H]-muscimol (20 Ci / mmol, D...

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Abstract

This invention relates to cation conducting GABAA receptors, mutated GABAA receptor subunits, polynucleotide sequences encoding mutated subunits, expression vectors comprising the mutated subunits, host cells capable of expressing the mutated subunits, drug screening methods, and chemical substances identified by the drug screening methods of the invention.

Description

[0001] This application is a Continuation of co-pending Application No. 10 / 479,251 filed on Dec. 1, 2003 and for which priority is claimed under 35 U.S.C. § 120. Application No. 10 / 479,251 is the national phase of PCT International Application No. PCT / DK02 / 00378 filed on Jun. 4, 2002 under 35 U.S.C. § 371. The entire contents of each of the above-identified applications are hereby incorporated by reference.TECHNICAL FIELD [0002] This invention relates to cation conducting GABAA receptors, mutated GABAA receptor subunits, polynucleotide sequences encoding mutated subunits, expression vectors comprising the mutated subunits, host cells capable of expressing the mutated subunits, drug screening methods, and chemical substances identified by the drug screening methods of the invention. BACKGROUND ART [0003] Preferred drug screening method of ion channels include the standard high throughput screens (HTS) using mixtures of test compounds and biological reagents along with indicator compo...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/00A61P25/00A61P37/08A61P9/00C07H21/04C07K14/435C07K7/00C12N15/11C12Q1/02C12N15/09A61K38/17A61K45/00A61P1/04A61P9/02A61P9/04A61P9/10A61P9/12A61P11/06A61P11/10A61P13/02A61P13/08A61P25/08A61P25/16A61P25/18A61P25/20A61P25/22A61P25/24A61P25/28A61P35/00A61P43/00C07K14/00C07K14/705C12N1/15C12N1/19C12N1/21C12N5/10
CPCC07K14/70571A61P1/04A61P11/06A61P11/10A61P13/02A61P13/08A61P25/00A61P25/08A61P25/16A61P25/18A61P25/20A61P25/22A61P25/24A61P25/28A61P35/00A61P37/08A61P43/00A61P9/00A61P9/02A61P9/04A61P9/10A61P9/12
Inventor JENSEN, MARIANNE LERBECHAHRING, PHILIP K.VARMING, THOMAS
Owner JENSEN MARIANNE LERBECH
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