PRIMARY STRUCTURE AND FUNCTIONAL EXPRESSION OF A CORTICAL COLLECTING DUCT K-IR CHANNEL

Maintenance of a negative membrane potential in the cortical collectin g duct (CCD) principal cell depends on a small-conductance, inward-rec tifying basolateral membrane K+ (K-ir) channel. In the present study, a candidate cDNA encoding this K+ channel, CCD-IRK3, was isolated from a mouse collecting duct cell line, M1. CCD-IRK3 shares a high degree of homology with a human brain inward-rectifier K+ channel (K-ir 2.3). By Northern analysis, CCD-IRK3 transcript (2.9 kb) was readily detect ed in M1 CCD cells but not in Madin-Darby canine kidney, LLC-PK1, Chin ese hamster ovary, or monkey kidney fibroblast cell lines. CCD-IRK3-sp ecific reverse transcription-polymerase chain reaction confirmed bonaf ide expression in the kidney. Functional expression studies in Xenopus oocytes revealed that CCD-IRK3 operates as strongly inward-rectifying K+ channel. The cation selectivity profile of CCD-IRK3 [ionic permeab ility values (P-K/P-i), Tl greater than or equal to Rb greater than or equal to K+ much greater than NH4 > Na; inward-slope conductance (G(K )/G(i)), Tl greater than or equal to K+ much greater than NH4 > Na > R b] is similiar to the macroscopic CCD basolateral membrane K+ conducta nce (G(K)/G(i) K+ much greater than NH4 > Rb; P-K/P-i, Rb approximate to K+ much greater than NH4). CCD-IRK3 also exhibits the pharmacologic al features of the native channel. Patch-clamp analysis reveals that C CD-IRK3 functions as a high open probability, voltage-independent, sma ll-conductance channel (14.5 pS), consistent with the native channel. Based on these independent lines of evidence, CCD-IRK3 is a possible c andidate for the small-conductance basolateral K-ir channel in the CCD .