CLONING SAD CHARACTERIZATION OF MULTIPLE FORMS OF THE HUMAN KIDNEY ROM-K POTASSIUM CHANNEL

The rat kidney ROM-K1 potassium channel cDNA was used to clone the hom olog from human kidney using a combination of cDNA cloning, reverse tr anscriptase-polymerase chain reaction (RT-PCR), and primer extension c loning methods. In addition to the human species homolog of ROM-K1, fo ur additional transcripts that are formed by alternative splicing of a single human gene were also characterized (hROM-K2 to hROM-K5). All f ive transcripts share a common 3' exon that encodes the majority of th e channel protein and in three of the isoforms translation is initiate d at a start codon contained within this exon (hROM-K2, hROM-K4, and h ROM-K5). The two other transcripts contain additional exons that poten tially extend the open reading frame by either 19 amino acid residues (hROM-K1) or by 17 amino acid residues (hROM-K3). Comparison of the tr anslation products from the three representative transcripts (hROM-K1, hROM-K2, and hROM-K3) confirmed that hROM-K1 gave the largest product (41.6 kDa) and was translated more efficiently than either hROM-K2 or hROM-K3. Also, despite the presence of several additional canonical a cceptor sites for Asn-linked glycosylation relative to rat ROM-K1, all three channel polypeptides were glycosylated to a similar extent in t he in vitro translation reactions when canine pancreatic microsomes we re included. A survey of the tissue distribution of expression of the various forms in selected human tissues showed that the core-exon link ed to all four possible 5' exons are detected almost exclusively in ki dney. The core-exon was also detected in human kidney and lower amount s were detected in skeletal muscle > pancreas > spleen > brain = heart > liver RNAs by RT-PCR. Alternatively, Northern blot analysis of poly (A)(+) RNAs from these same tissues revealed a 2.8-kilobase transcript only in kidney. Heterologous expression of either the hROM-K1, hROM-K 2, or hROM-K3 channel transcripts in Xenopus oocytes led to the expres sion of K+-selective, Ba2+-sensitive inwardly rectifying channels as m easured by whole cell currents. At this level of analysis, the channel properties of the individual forms could not be distinguished.