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Am J Physiol Heart Circ Physiol 278: H1153-H1162, 2000;
0363-6135/00 $5.00
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Vol. 278, Issue 4, H1153-H1162, April 2000

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

Lei Liu1,*, Q. Ivy Fan1,*, Mohamad R. El-Zaru1, Kathleen Vanderpool1, Ronald N. Hines2, and James D. Marsh1

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 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 alpha 1C-subunit gene, we have cloned a 2.3-kb fragment containing the 5'-flanking sequences and identified the alpha 1C-subunit gene transcription start site. The rat alpha 1C-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.

calcium channel; promoter; transcription; myocyte; heart; dihydropyridine

*  L. Liu and Q. I. Fan contributed equally to this work.




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