CLONING AND CHARACTERIZATION OF 2 K-IR)1.1 POTASSIUM CHANNEL HOMOLOGSFROM HUMAN KIDNEY (K(IR)1.2 AND K(IR)1.3)( INWARD RECTIFIER (K)

The DNA sequence encoding the rat brain inward rectifier-10 K+ channel was amplified from rat brain RNA using reverse transcription-polymera se chain reaction and used to clone the human homolog. Low stringency screening of a human kidney cDNA library and subsequent DNA sequence a nalysis 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 dis tinct human genes, K(ir)1.2 represents the human homolog of the rat BI RK-10 sequence, whereas K(ir)1.3 was unique compared with all availabl e sequence data bases, The genes that encode K(ir)1.2 and K(ir)1.3 wer e mapped to human chromosomes 1 and 21, respectively. Both genes showe d tissue-specific expression when analyzed by Northern blots, K(ir)1.2 was only detected in brain much greater than kidney and was detected at high levels in all brain regions examined, K(ir)1.3 was most readil y detected in kidney and was also expressed in pancreas > lung, Compar ative 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 differe nce between the two polypeptides is that the Walker Type A consensus b inding motif present in both K(ir)1.1 and K(ir)1.2 was not conserved i n the K(ir)1.3 sequence. Expression of the K(ir)1.2 polypeptide in Xen opus 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 red uced by >85% when the pH was decreased from pH 7.4 to 5.9 using the me mbrane-permeant buffer acetate but was relatively unaffected when pH w as similarly lowered using membrane impermeant biphthalate, The inhibi tion by intracellular protons was voltage-independent with an IC50 of pH 6.2 and a Hill coefficient of 1.9, suggesting the cooperative bindi ng 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 polypeptid e of the expected M(r) in an in vitro translation system. Go-expressio n of K(ir)1.3 with either K(ir)1.1 or K(ir)1.2 reduced currents result ing from expression of these inward-rectifier subunits alone, consiste nt with a dominant negative influence on K(ir)1.1 and K(ir)1.2 express ion.