Characterization of the cardiac KCNE1 gene promoter.

BACKGROUND

KCNE1 encodes an essential cardiac slow delayed-rectifier potassium current (I(Ks)) beta-subunit (minK). Varying minK expression is important in disease-related remodeling and species-dependent expression. This study addressed 5'-regulatory elements that potentially control KCNE1 transcription.

METHODS AND

RESULTS

The transcriptional start site of human KCNE1 (HKCNE1) was determined with 5'RACE. Of four isoforms, the putative promoter driving the isoforms constituting >80% expression in human hearts was further analyzed. A 1625-bp region 5' to the transcriptional start site was subcloned into luciferase-reporter plasmid (PGL3-Basic). The full promoter sequence increased luciferase expression 31-fold in neonatal rat cardiomyocytes (NRMs). A much smaller 327-bp core promoter maintained activity 21-29 fold. The core promoter conferred cardiomyocyte-preferential expression, with an activity in NRMs 4.9-fold greater than in Chinese Hamster Ovary cells (CHOs), compared to approximately 2.0 for the full-length promoter. Site-directed mutagenesis of all three GATA elements in the core promoter reduced its activity by >50% and attenuated cardiomyocyte-preferential expression. Mutagenesis of the second GATA element alone decreased promoter activity by approximately 50%. GATA4 knockdown with siRNA inhibited approximately 40% of core promoter activity in NRMs. Angiotensin-II increased HKCNE1 promoter activity, but only in the presence of intact GATA elements. The typically low-level I(Ks) expression in mouse and rabbit is related to low minK expression. Cloning of the mouse KCNE1 (MKCNE1) 5'-regulatory region showed approximately 50% sequence identity to human. MKCNE1 had only 1 GATA element in the region corresponding to the human core promoter and had less promoter activity (11.7 vs 29.0-fold PGL3-Basic for human).

CONCLUSION

Promoter elements in the HKCNE1 5'-end, particularly GATA binding sites, may be important in tissue, disease and species-related transcriptional regulation of I(Ks).