Purified and isolated potassium-chloride cotransporter nucleic acids and polypeptides and therapeutic and screening methods using same

Abstract

KCC2, KCC3, and KCC4 potassium-chloride cotransporter proteins, and nucleic acid molecules encoding the same. Recombinant host cells, recombinant nucleic acids and recombinant proteins are also disclosed, along with methods of producing each. Isolated and purified antibodies to KCC2, KCC3, and KCC4 homologs, and methods of producing the same, are also disclosed. KCC2, KCC3, and KCC4 gene products have biological activity in potassium-chloride cotransport. Thus, therapeutic methods involving this activity are also disclosed.

1 . An isolated and purified, biologically active KCC3 potassium-chloride cotransporter polypeptide comprising: 
 (a) a polypeptide encoded by a nucleic acid sequence as set forth in any of SEQ ID NOs: 3, 5, 7, 9, and 15;    (b) a polypeptide encoded by a nucleic acid sequence having 75% or greater sequence identity to nucleotides 1-434 of SEQ ID NOs: 3, 5, 7, 9, and 15;    (c) a polypeptide having an amino acid sequence as set forth in SEQ ID NOs: 4, 6, 8, 10, and 16;    (d) a polypeptide which is a biological equivalent of the polypeptide set forth in SEQ ID NOs: 4, 6, 8, 10, and 16;    (e) a polypeptide which is immunologically cross-reactive with an antibody which is immunoreactive with a polypeptide comprising part or all of the first 90 amino acids of any SEQ ID NOs: 4, 6, 8, 10, and 16; or    (f) a polypeptide encoded by a nucleic acid molecule capable of hybridizing under stringent conditions to a nucleic acid molecule comprising the first 434 nucleotides of any of SEQ ID NOs: 3, 5, 7, 9, and 15, or complement thereof.    
     
     
         2 . The polypeptide of  claim 1 , wherein the polypeptide comprises a mouse KCC3 potassium-chloride cotransporter polypeptide or a human KCC3 potassium-chloride cotransporter polypeptide.  
     
     
         3 . The polypeptide of  claim 1 , modified to be in detectably labeled form.  
     
     
         4 . An isolated and purified antibody capable of specifically binding to the polypeptide of  claim 1 .  
     
     
         5 . The antibody of  claim 4 , wherein the antibody is capable of modulating the biological activity of the polypeptide of  claim 1 .  
     
     
         6 . A hybridoma cell line which produces the antibody of  claim 5 .  
     
     
         7 - 57 . (canceled)  
     
     
         58 . An assay kit for detecting the presence, in a biological sample, of an antibody immunoreactive with a potassium-chloride cotransporter polypeptide, the kit comprising a polypeptide of  claim 1 , that immunoreacts with the antibody, with the polypeptide present in an amount sufficient to perform at least one assay.  
     
     
         59 - 75 . (canceled)  
     
     
         76 . A pharmaceutical composition comprising a therapeutically effective amount of a modulator of a biological activity of KCC2, KCC3, KCC4, or combinations thereof, and a pharmaceutically acceptable diluent or vehicle.  
     
     
         77 . The pharmaceutical composition of  claim 76 , wherein the KCC2-, KCC3- and/or KCC4-biological-activity-modulator is selected from the group consisting of: 
 (a) a purified antibody which preferentially binds KCC2, KCC3, KCC4, combinations thereof, or a fragment or derivative thereof, and    (b) a polypeptide which interacts KCC2, KCC3, KCC4, combinations thereof, or a fragment or derivative thereof.    
     
     
         78 - 99 . (canceled)