ROMK INWARDLY RECTIFYING ATP-SENSITIVE K+ CHANNEL .2. CLONING AND DISTRIBUTION OF ALTERNATIVE FORMS

The rat ROMK gene encodes inwardly rectifying, ATP-regulated K+ channe ls [K. Ho, C. G. Nichols, W. J. Lederer, J. Lytton, P. M. Vassilev, M. V. Kanazirska, and S. C. Hebert. Nature Lend. 362: 31-38, 1993; H. Zh ou, S. S. Tate, and L. G. Palmer. Am. J. Physiol. 266 (Cell Physiol. 3 5): C809-C824, 1994], and mRNA encoding these channels is widely expre ssed in distal cortical and outer medullary nephron segments [see comp anion study; W.-S. Lee and S. C. Hebert. Am. J. Physiol. 268 (Renal Fl uid Electrolyte Physiob. 37): F1124-F1131, 1995]. Using approaches bas ed on homology to ROMK1, we have identified two additional ROMK isofor ms, ROMK2b and ROMK3. Analysis of the nucleotide sequences of the ROMK isoforms indicates that molecular diversity of ROMK transcripts is du e to alternative splicing at both the 5'-coding and 3'-noncoding regio ns. The splicing at the 5' end of ROMK gives rise to channel proteins with variable-length NH2, termini containing different initial amino a cid sequences. Functional expression of these isoforms in Xenopus oocy tes showed that they form functional Ba2+-sensitive K+ channels. The n ephron distribution of mRNAs encoding alternatively spliced isoforms o f ROMK (ROMK1-ROMK3) was investigated by reverse transcription-polymer ase chain reaction (RT-PCR) of nephron segments dissected from rat kid ney. Nondegenerate PCR primer pairs were designed to span at least one intron and to amplify specific alternatively spliced forms of ROMK. T he validities of PCR-amplified products were verified by nucleotide se quencing. ROMK1-specific PCR product was found only in the collecting duct (both the cortical and outer medullary collecting duct), while th e ROMK2b-specific product was found in all nephron segments from medul lary thick ascending limb to the cortical collecting duct. ROMK3-speci fic product was identified only in medullary and cortical thick ascend ing Limb and distal convoluted tubule. Thus expression of the ROMK iso form with the shortest NH2-terminal region, ROMK2b, is widely distribu ted along the loop of Henle and distal nephron, while the spliced form s with the longer NH2 termini, ROMK1 and ROMK3, are differentially exp ressed in this region of the nephron. The potential significance of th e differential distribution of these ROMK isoforms is discussed.