Adenoviral-mediated expression of dihydropyridine-insensitive L-type calcium channels in cardiac ventricular myocytes and fibroblasts.
Cardiac voltage-gated Ca2+ channels regulate the intracellular Ca2+ concentration and are therefore essential for muscle contraction, second messenger activation, gene expression and electrical signaling. As a first step in accessing the structural versus functional properties of the L-type Ca2+ channel in the heart, we have expressed a dihydropyridine (DHP)-insensitive CaV1.2 channel in rat ventricular myocytes and fibroblasts. Following isolation and culture, cells were infected with adenovirus expressing either LacZ or a mutant CaV1.2 channel (CaV1.2DHPi) containing the double mutation (T1039Y & Q1043M). This mutation renders the channel insensitive to neutral DHP compounds such as nisoldipine. The whole-cell, L-type Ca2+ current (ICa) measured in control myocytes was inhibited in a concentration-dependent manner by nisoldipine with an IC50 of 66 nM and complete block at 250 nM. In contrast, ICa in cells infected with AdCaV1.2DHPi was inhibited by only 35% by 500 nM nisoldipine but completely blocked by 50 microM diltiazem. In order to study CaV1.2DHPi in isolation, myocytes infected with AdCaV1.2DHPi were incubated with nisoldipine. Under this condition the cells expressed a large ICa (12 pA/pF) and displayed Ca2+ transients during field stimulation. Furthermore, addition of 2 microM forskolin and 100 microM 3-isobutyl-1-methylxanthine (IBMX), to stimulate protein kinase A, strongly increased IBa in the AdCaV1.2DHPi-infected cells. A Cd2+-sensitive IBa was also recorded in cardiac fibroblasts infected with AdCaV1.2DHPi. Thus, expression of CaV1.2DHPi will provide an important tool in studies of cardiac myocyte and fibroblast function.
Walsh
Kenneth B
KB
Department of Pharmacology, Physiology & Neuroscience, University of South Carolina, School of Medicine, Columbia, SC 29208, United States. walsh@med.sc.edu
Zhang
Jining
J
Fuseler
John W
JW
Hilliard
Nathan
N
Hockerman
Gregory H
GH
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D004110:Diltiazem / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000378:metabolism*; D006352:Heart Ventricles; D009200:Myocardial Contraction / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000378:metabolism*; D015737:Nisoldipine / Q000494:pharmacology*; D051381:Rats
D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D015737:Nisoldipine; D017868:Cyclic AMP-Dependent Protein Kinases; D004110:Diltiazem; D002118:Calcium