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1 Program in Molecular and Cellular Cardiology and 2 Departments of Medicine and Pharmacology, Harper Hospital, Wayne State University School of Medicine, and Department of Veterans Affairs Medical Center, Detroit, Michigan 48201
The
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 1C-subunit gene, we have cloned a 2.3-kb
fragment containing the 5'-flanking sequences and identified the
1C-subunit gene transcription start site. The rat
1C-subunit gene promoter belongs to the TATA-less class of such basal elements. Using deletion analysis of
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
1C-subunit gene. Electrophoretic mobility shift assays identified a region of the 1C-subunit gene promoter that
can bind transcription factors and appears to be important for gene expression.
calcium channel; promoter; transcription; myocyte; heart; dihydropyridine
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