BASOLATERAL MEMBRANE TARGETING OF A RENAL-EPITHELIAL INWARDLY RECTIFYING POTASSIUM CHANNEL FROM THE CORTICAL COLLECTING DUCT, CCD-IRK3, IN MDCK CELLS

We recently cloned an inward-rectifying K channel (Kir) cDNA, CCD-IRK3 (mKir 2.3), from a cortical collecting duct (CCD) cell line, Although this recombinant channel shares many functional properties with the ' 'small-conductance'' basolateral membrane Kir channel in the CCD, its precise subcellular localization has been difficult to elucidate by co nventional immunocytochemistry. To circumvent this problem, we studied the targeting of several different epitope-tagged CCD-IRK3 in a polar ized renal epithelial cell line, Either the Il-amino acid span of the vesicular stomatitis virus (VSV) G glycoprotein (P5D4 epitope) or a 6- amino acid epitope of the bovine papilloma virus capsid protein (AU1) was genetically engineered on the extreme N terminus of CCD-IRK3, As d etermined by patch-clamp and two-microelectrode voltage-clamp analyses in Xenopus oocytes, neither tag affected channel function; no differe nces in cation selectivity, barium block, single channel conductance, or open probability could be distinguished between the wild-type and t he tagged constructs, MDCK cells were transfected with tagged CCD-IRK3 , and several stable clonal cell lines were generated by neomycin-resi stance selection, Immunoprecipitation studies with anti-PSD? or anti-A U1 antibodies readily detected the predicted-size 50-kDa protein in th e transfected cells lines but not in wild-type or vector-only (PcB6) t ransfected MDCK cells, As visualized by indirect immunofluorescence an d confocal microscopy, both the tagged CCD-IRK3 forms were exclusively detected on the basolateral membrane, To assure that the VSV G tag wa s not responsible for the targeting, the P5D4 epitope modified by a si te-directed mutagenesis (Y2F) to remove a potential basolateral target ing signal contained in this tag, VSV(Y2F) was also detected exclusive ly on the basolateral membrane, confirming bonafide IRK3 basolateral e xpression, These observations, with our functional studies, suggest th at CCD-IRK3 may encode the small-conductance CCD basolateral K channel .