Isolation of putative voltage-gated epithelial K-channel isoforms from rabbit kidney and LLC-PK1 cells.
Epithelial voltage-gated potassium (K) channels have been well studied using electrophysiological methods, but little is known about their structures. We tested the hypothesis that some of these channels belong to the Shaker gene family, which encodes voltage-gated K channels in excitable tissues. From published sequences of Shaker proteins in Drosophila, rat, and mouse brain, we chose regions that were conserved between species. Based on these protein sequences, degenerate oligonucleotides flanking the putative voltage sensor (S4) were synthesized and used as primers for the polymerase chain reaction. Five Shaker-like cDNAs were amplified from rabbit kidney cortex and three from LLC-PK1, an epithelial cell line derived from pig kidney. Each partial-length rabbit kidney cDNA is approximately 850 base pairs (bp) long. The deduced amino acid sequences contain five putative transmembrane segments and are 79-97% identical to two Shaker isoforms expressed in rat brain (RBK1 and RBK2). Sequence similarity is greatest in the putative transmembrane segments S1-S5. Importantly, the S4 segment, the putative voltage gate is highly conserved in all 5 cDNAs. Southern analysis of rabbit genomic DNA suggests that each isoform is encoded by a different gene. The partial length LLC-PK1 cDNAs are 450-bp long, and the deduced amino acid sequences are 77-99% identical to the rabbit cDNAs. This is, to our knowledge, the first demonstration that Shaker-like genes are expressed in renal epithelial cells. These genes most likely encode voltage-gated K channels involved in renal epithelial K transport.
Desir
G V
GV
Department of Medicine, Yale University School of Medicine, New Haven 06510.
Hamlin
H A
HA
Puente
E
E
Reilly
R F
RF
Hildebrandt
F
F
Igarashi
P
P
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004330:Drosophila / Q000235:genetics; D004594:Electrophysiology; D004848:Epithelium / Q000378:metabolism; D015870:Gene Expression; D015640:Ion Channel Gating / Q000502:physiology*; D007536:Isomerism; D007668:Kidney / Q000378:metabolism*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011817:Rabbits
D015345:Oligonucleotide Probes; D015221:Potassium Channels