Abstract 1096: The Cardiac KCNE1 Gene Promoter and Transcriptional Regulation of IKs Expression
Introduction: The KCNE1 gene encodes a β-subunit (minK) that is essential for cardiac slow delayed rectifier K+ current (IKs) formation. There is evidence that variations in KCNE1 expression may be important in disease-related IKs remodeling and species-dependent expression. This study addressed the 5′-regulatory elements that may contribute to this regulation.
Methods and Results: The transcription start site of the human KCNE1 (HKCNE1) gene was determined and three isoforms identified. The isoform constituting >80% expression in human heart was selected for further analysis. A 1625 bp region 5′ to the transcriptional start site (full-length promoter, FLP) was cloned and promoter activity analyzed by subcloning into luciferase reporter plasmid followed by expression in neonatal rat cardiomyocytes (NRMs) and Chinese Hamster ovary (CHO) cells. Successive deletions showed that the core promoter (CP: -311/+16; 327-bp) retains cardiomyocyte-specific expression (NRM/CHO expression ratio=4.9 vs ~2.0 for FLP) and promoter activity (20.7±2.2 vs 31.7±6.3 fold PLG3-basic for FLP). Site-directed mutagenesis of three GATA boxes in CP reduced promoter activity and cardiac-specific expression, with mutation of the 2nd GATA being sufficient to strongly reduce both (activity to 12.9±0.9 fold PLG3-basic, NRM/CHO ratio to 2.3). Suppression of GATA4 expression by siRNA decreased promoter activity to 16.2±1.0 fold PLG3-basic, P<0.001 vs CP. KCNE1 is important for species-specific IKs expression, with low minK levels in the mouse associated with very small IKs. We cloned the mouse KCNE1 5′-regulatory region corresponding to the human CP. The mouse CP lacked the 3 GATA boxes found in its human counterpart, showing only 14.6±1.6 fold PGL3 basic activity and a NRM/CHO ratio of 3.0, both significantly less than human. Angiotensin II, which may be responsible for disease-induced IKs upregulation, increased activity of the human CP (eg from 31.0±4.3 to 40.4±0.6 fold at 1 μM, P<0.05), an effect eliminated by mutation of the 2nd GATA box.
Conclusions: Promoter elements 5′ to the KCNE1 TSS, and in particular GATA boxes in the core promoter, are important in transcriptional regulation, cardiac-specificity, disease-related changes and interspecies variation of IKs.