Cloning and characterization of two K+ inward rectifier (K(ir)) 1.1 potassium channel homologs from human kidney (K(ir)1.2 and K(ir)1.3)

Abstract

The DNA sequence encoding the rat brain inward rectifier-10 K+ channel was amplified from rat brain RNA using reverse transcription-polymerase chain reaction and used to clone the human homolog. Low stringency screening of a human kidney cDNA library and subsequent DNA sequence analysis identified two related K+ inward rectifier cDNAs, referred to as K(ir)1.2 and K(ir)1.3, which were derived from transcription of distinct human genes. K(ir)1.2 represents the human homolog of the rat BIRK-10 sequence, whereas K(ir)1.3 was unique compared with all available sequence data bases. The genes that encode K(ir)1.2 and K(ir)1.3 were mapped to human chromosomes 1 and 21, respectively. Both genes showed tissue-specific expression when analyzed by Northern blots. K(ir)1.2 was only detected in brain >> kidney and was detected at high levels in all brain regions examined. K(ir)1.3 was most readily detected in kidney and was also expressed in pancreas > lung. Comparative analysis of the predicted amino acid sequences for K(ir)1.2 and K(ir)1.3 revealed they were 62% identical. The most remarkable difference between the two polypeptides is that the Walker Type A consensus binding motif present in both K(ir)1.1 and K(ir)1.2 was not conserved in the K(ir)1.3 sequence. Expression of the K(ir)1.2 polypeptide in Xenopus oocytes resulted in the synthesis of a K+-selective channel that exhibited an inwardly rectifying current-voltage relationship and was inhibited by external Ba2+ and Cs+. K(ir)1.2 current amplitude was reduced by >85% when the pH was decreased from pH 7.4 to 5.9 using the membrane-permeant buffer acetate but was relatively unaffected when pH was similarly lowered using membrane- impermeant biphthalate. The inhibition by intracellular protons was voltage- independent with an IC50 of pH 6.2 and a Hill coefficient of 1.9, suggesting the cooperative binding of 2 protons to the intracellular face of the channel. In contrast, K(ir)1.3 expression in Xenopus oocytes was not detectable despite the fact that the cRNA efficiently directed the synthesis of a polypeptide of the expected M(r) in an in vitro translation system. Co- expression of K(ir)1.3 with either K(ir)1.1 or K(ir)1.2 reduced currents resulting from expression of these inward-rectifier subunits alone, consistent with a dominant negative influence on K(ir)1.1 and K(ir) 1.2 expression.