Cloning and characterization of multiple forms of the human kidney ROM-K potassium channel

Abstract

The rat kidney ROM-K1 potassium channel cDNA was used to clone the homolog from human kidney using a combination of cDNA cloning, reverse transcriptase-polymerase chain reaction (RT-PCR), and primer extension cloning methods. In addition to the human species homolog of ROM-K1, four additional transcripts that are formed by alternative splicing of a single human gene were also characterized (hROM-K2 to hROM-K5). All five transcripts share a common 3' exon that encodes the majority of the channel protein and in three of the isoforms translation is initiated at a start codon contained within this exon (hROM-K2, hROM-K4, and hROM-K5). The two other transcripts contain additional exons that potentially extend the open reading frame by either 19 amino acid residues (hROM-K1) or by 17 amino acid residues (hROM-K3). Comparison of the translation 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 acceptor sites for Asn-linked glycosylation relative to rat ROM-K1, all three channel polypeptides were glycosylated to a similar extent in the in vitro translation reactions when canine pancreatic microsomes were included. A survey of the tissue distribution of expression of the various forms in selected human tissues showed that the core-exon linked to all four possible 5' exons are detected almost exclusively in kidney. The core-exon was also detected in human kidney and lower amounts 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-K2, or hROM-K3 channel transcripts in Xenopus oocytes led to the expression of K(+)-selective, Ba(2+)-sensitive inwardly rectifying channels as measured by whole cell currents. At this level of analysis, the channel properties of the individual forms could not be distinguished.