Cation conducting gabaa receptors and their use

Abstract

This invention relates to cation conducting GABA A receptors, mutated GABA A 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.

1 . A cation-conducting human GABA A  receptor.  
     
     
         2 . The receptor of  claim 1 , comprising at least one GABA A  receptor subunit, which subunit holds one or more mutations in the loop bridging its first and its second transmembrane domain (TM1 and TM2).  
     
     
         3 . The receptor of  claim 2 , which has-been mutated at amino acid positions −6′ to −1′ when numbered according to TM2.  
     
     
         4 . The receptor of  claim 3 , comprising at least one mutated α subunit, and/or at least one mutated β subunit, and/or at least one mutated γ subunit.  
     
     
         5 . The receptor of  claim 4 , comprising at least one mutated α1, α2, α3, α4, α5, α6, β1, β2, β3, γ1 γ2 and/or γ3 subunit.  
     
     
         6 . A mutated GABA A  receptor subunit, comprising one or more mutations in the loop bridging its first and second transmembrane domain (TM1 and TM2).  
     
     
         7 . The receptor subunit of  claim 6 , which has been mutated at amino acid positions −6′ to −1′ when numbered according to TM2.  
     
     
         8 . The receptor subunit of either of claims  6 -7, being a mutated α1, α2, α3, α4, α5, α6, β1, β2, β3, γ1 γ2 or γ3 subunit.  
     
     
         9 . The receptor subunit of any of  claim 6 , holding the sequence X 1 X 2 X 3 X 4 X 5  (SEQ ID NO: 18); 
 wherein 
 X 1  designates D or E;  
 X 2  designates S, C or A;  
 X 3  designates G. A or V;.  
 X 4  designates. E or D;. and  
 X 5  designates K or R.  
   
     
     
         10 . The receptor subunit of any of  claim 6 , holding the sequence DSGEK (SEQ ID NO: 17) or a subsequence thereof.  
     
     
         11 . The receptor subunit of  claim 10 , holding a sequence selected from the sequences GE, SGE, DSGE (SEQ ID NO: 55), GEK, SGEK (SEQ ID NO: 56), DSGEK (SEQ ID NO: 17), DS, DSG, EK, and GEK.  
     
     
         12 . A polynucleotide sequence encoding the mutated subunit of  claim 6 .  
     
     
         13 . The polynucleotide sequence of  claim 12 , encoding a mutated α1, α2, α3, α4, α5, α6, β1, β2, β3, γ1 γ2 and/or γ3 subunit.  
     
     
         14 . An expression vector comprising the polynucleotide sequence of  claim 12 .  
     
     
         15 . A host cell comprising the polynucleotide sequence of  claim 12 , or the expression vector of  claim 14 .  
     
     
         16 . A method of screening a chemical compound for inhibiting, activating or modulating activity of a cation-conducting GABA A  receptor, which method comprises the steps of 
 (i) subjecting a cation-conducting GABA A  receptor containing cell to the action of the chemical compound to be screened;    (ii) subjecting the cation-conducting GABA A  receptor containing cell to activation with GABA or any other GABA-acting substance; and    (iii) monitoring ion flux through the cation-conducting GABA A receptor, either directly or indirectly, and thereby determining the action of the chemical compound.    
     
     
         17 . The method of  claim 16 , wherein the cation-conducting GABA A  receptor containing cell is a HEK293 cell, a CHO-k1 cell, a BHK cell, a COS7 cell, a PC12 cell, a HiBS cell, a RN33b cell, or a Xenopus laevis oocyte (XLO), or any other cell line able to express the cation-conducting GABA A  receptor.  
     
     
         18 . The method of either of claims  16 - 17 , wherein monitoring of the ion flux of the cation-conducting GABA A  receptor is performed using fluorescence or radio-ligand methods.  
     
     
         19 . The method of  claim 16 , wherein the cation-conducting GABA A  receptor-containing cell is loaded or incubated with a fluorescence indicator or a radio-ligand, that allows for a determination of changes in ion flux through the cation-conducting GABA A  receptor.  
     
     
         20 . The method of  claim 19 , wherein the fluorescent indicator is FLUO-3, FLUO-4, Calcium Green, FURA-2, SBFI, PBFI, CD222, BCECF, DIBAC 4 (3), DiOC5(3) or DiOC2(3).  
     
     
         21 . The method of  claim 19  wherein the radio-ligand is Rb + or an organic cation such as TPP   + .  
     
     
         22 . The method of  claim 16 , wherein monitoring of the ion flux of the cation-conducting GABA A  receptor is performed by spectroscopic methods, e.g. using a FLIPR assay (Fluorescence Image Plate Reader, available from Molecular Devices).  
     
     
         23 . The method of  claim 16 , wherein monitoring of the ion flux of the cation-conducting GABA A  receptor is performed by patch clamp techniques.  
     
     
         24 . A chemical compound identified according to the method of  claim 16 .  
     
     
         25 . Use of the chemical compound identified according to  claim 24  for diagnosis, treatment, prevention or alleviation of a disease or a disorder or a condition of a mammal, including a human, which disease, disorder or condition is related to GABA A  receptor dysfunction.  
     
     
         26 . The use according to  claim 25 , wherein the disease, disorder or condition is asthma, acute heart failure, hypotension, urinary retention, osteoporosis, hypertension, angina pectoris, myocardial infarction, ulcers, allergies, benign prostatic hypertrophy, prostate cancer, Parkinson's disease, psychotic and neurological disorders, anxiety, schizophrenia, mania, depression, dyskinesia, memory disorders, sleep disorders, convulsive disorders, and epilepsy.