Genomic structure and regulation of Kcn1, a cGMP-gated potassium channel

We recently cloned a novel rabbit gene that encodes a 725-amino acid protein (Kcn1) (Y. Yao, A. S. Segal, P. Welling, X. Zhang, C. M. McNicholas, D. Engel, E. L. Boulpaep, and G. Desir. Proc. Natl. Acad. Sci. USA 92: 11711-11715, 1995). Kcn1 RNA injected in Xenopus oocytes leads to the expression of potassium channels that are specifically activated by guanosine 3',5'-cyclic monophosphate (cGMP). Northern blot and ribonuclease (RNase) protection analysis show that Kcn1 is differentially expressed in kidney, aorta, brain, and heart. The purpose of present study is to determine the structure of Kcn1 gene, analyze the promoter region, and identify cis-regulatory elements responsible for transcription. We find that the coding region of Kcn is intronless. The major transcription initiation site was identified by primer extension. Sequence analysis of the 5'-flanking region indicates that, although the gene lacks a typical TATA box, it does have a TATA-box-like region (-TAT-). Using luciferase reporter constructs transfected in the porcine kidney cell line (LLC-PK1), the promoter region and a 5' enhancer element were identified by deletion analysis. Phorbol esters (12-O-tetradecanoylphorbol-13-acetate) and forskolin stimulated Kcn1 gene expression 2.5- and 3.5-fold, respectively. In conclusion, we have identified the region of the novel potassium channel gene, Kcn1, that contains the promoter, a 5' enhancer, and several cis-regulatory elements and shown that gene transcription is stimulated by cAMP and phorbol esters.