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 dihydropyridine (DHP)-binding site (the DHP receptor), provides the pore structure for Ca2+ entry. It contains the binding sites for multiple classes of drugs collectively known as Ca2+ antagonists. As an initial step toward understanding the mechanisms controlling transcription of the rat cardiac alc-subunit gene, we have cloned a 2.3-kb fragment containing the 5'-flanking sequences 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 modulating activity of the 5'-flanking region and conducted transient transfections in cultured neonatal rat cardiac ventricular myocytes and vascular smooth muscle cells. Sequence scanning identified several potential regulatory elements, including five consensus sequences for the cardiac-specific transcription factor Nkx2.5, an AP-1 site, a cAMP response element, and a hormone response element. Transient transfection experiments with the promoter-luciferase reporter fusion gene demonstrate that the 2-kb 5'-flanking region confers tissue specificity and hormone responsiveness to expression of the Ca2+ channel alpha(1C)-subunit gene. Electrophoretic mobility shift assays identified a region of the alpha(1C)-subunit gene promoter that can bind transcription factors and appears to be important for gene expression.