title: 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.
authors.affiliation: Department of Cell Biology, Upjohn Laboratories, Kalamazoo, Michigan 49007.
authors.forename: M E
authors.initials: ME
authors.lastname: Shuck
authors.forename: J H
authors.initials: JH
authors.lastname: Bock
authors.forename: C W
authors.initials: CW
authors.lastname: Benjamin
authors.forename: T D
authors.initials: TD
authors.lastname: Tsai
authors.forename: K S
authors.initials: KS
authors.lastname: Lee
authors.forename: J L
authors.initials: JL
authors.lastname: Slightom
authors.forename: M J
authors.initials: MJ
authors.lastname: Bienkowski
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000235:genetics* / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying; D012326:RNA Splicing; D051381:Rats; D014018:Tissue Distribution; D014981:Xenopus
chemical_list: D018076:DNA, Complementary; C086989:KCNJ1 protein, human; C510692:Kcnj1 protein, rat; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying