The romk inwardly rectifying atp-sensitive k channel. ii. cloning and intra-renal distribution of alternatively spliced forms.. Boim, Mirian A., M. A., Kevin Ho, Mary E. Shuck, Michael J. Bienkowski, Jeff H. Block, Jerry L. Slightom, Yinhaiyang, Barry M. Brenner, and Steven C. Hebert. Laboratory of Molecular Physiology and Biophysics, Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, 75 Francis Street, Boston, MA 02115 and Departments of Cell Biology and Molecular Biology, Upjohn Laboratories, Kalamazoo, MI 49007
APStracts 2:0005F, 1995.
The rat ROMK gene encodes inwardly rectifying, ATP-regulated K+ channels (Ho, K.H., C. G. Nichols, W.J. Lederer, J. Lytton, P.M. Vassilev, M.V. Kanazirska, and S.C. Hebert. Nature Lond. 362:31-37, 1993; Zhou, H., S.S. Tate, and L.G. Palmer. Am. J. Physiol. 266:C809-C824, 1994) and mRNA encoding these channels is widely expressed in distal cortical and outer medullary nephron segments (see companion paper; Lee and Hebert. Am. J. Physiol.). Using approaches based on homology to ROMK1, we have identified two additional ROMK isoforms, ROMK2b and ROMK3. Analysis of the nucleotide sequences of the ROMK isoforms indicates that molecular diversity of ROMK transcripts is due to alternative splicing at both the 5'-coding and 3'-non-coding regions. The splicing at the 5' end of ROMK gives rise to channel proteins with varying length NH2-termini containing different initial amino acid sequences. Functional expression of these isoforms in Xenopus oocytes showed that they form functional Ba2+ -sensitive K+ channels. The nephron distribution of mRNAs encoding alternatively spliced isoforms of ROMK (ROMK1-ROMK3) was investigated by RT -PCR of nephron segments dissected from rat kidney. Non-degenerate PCR primer pairs were designed to span at least one intron and to amplify specific alternatively spliced forms of ROMK. The validity of PCR amplified products were verified by nucleotide sequencing. ROMK1-specific PCR product was found only in the collecting duct, both cortical and outer medullary collecting duct, while the ROMK2b-specific product was found in all nephron segments from medullary thick ascending limb to the cortical collecting duct. ROMK3 -specific product was identified only in medullary and cortical thick ascending limb and distal convoluted tubule. Thus, expression of the ROMK isoform with the shortest NH2-terminal region, ROMK2b, is widely distributed along the loop of Henle and distal nephron while the spliced forms with the longer NH2-termini, ROMK1 and ROMK3, are differentially expressed in this region of the nephron. The potential significance of the differential distribution of these ROMK isoforms is discussed. 

Received 11 November 1994; accepted in final form 4 January 1995.
APS Manuscript Number F363-4.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 23 February 1995.