Regulation of DHP receptor expression by elements in the 5 '-flanking sequence

The alpha(1)-subunit of the cardiac/vascular Ca2+ channel, which is the dih ydropyridine (DHP)-binding site (the DHP receptor), provides the pore struc ture for Ca2+ entry. It contains the binding sites for multiple classes of drugs collectively known as Ca2+ antagonists. As an initial step toward und erstanding the mechanisms controlling transcription of the rat cardiac alc- subunit gene, we have cloned a 2.3-kb fragment containing the 5'-flanking s equences and identified the alc-subunit gene transcription start site. The rat alc-subunit gene promoter belongs to the TATA-less class of such basal elements. Using deletion analysis of alpha(1C)-subunit promoter-luciferase reporter gene constructs, we have characterized the transcriptional modulat ing activity of the 5'-flanking region and conducted transient transfection s in cultured neonatal rat cardiac ventricular myocytes and vascular smooth muscle cells. Sequence scanning identified several potential regulatory el ements, including five consensus sequences for the cardiac-specific transcr iption factor Nkx2.5, an AP-1 site, a cAMP response element, and a hormone response element. Transient transfection experiments with the promoter-luci ferase reporter fusion gene demonstrate that the 2-kb 5'-flanking region co nfers tissue specificity and hormone responsiveness to expression of the Ca 2+ channel alpha(1C)-subunit gene. Electrophoretic mobility shift assays id entified a region of the alpha(1C)-subunit gene promoter that can bind tran scription factors and appears to be important for gene expression.