L-type calcium channels in insulin-secreting cells: Biochemical characterization and phosphorylation in RINm5F cells

Opening of dihydropyridine-sensitive voltage-dependent L-type Ca2+-channels (LTCCs) represents the final common pathway for insulin secretion in pancreatic p-cells and related cell lines. In insulin-secreting cells their exact subunit composition is unknown. We therefore investigated the subunit structure of (+)-[H-3]isradipine-labeled LTCCs in insulin-secreting RINm5F cells. Using subunit-specific antibodies we demonstrate that alpha 1C subunits (199 kDa, short form) contribute only a minor portion of the total alpha 1 immunoreactivity in membranes and partially purified Ca2+-channel preparations. However, alpha 1C forms a major constituent of (+)-[H-3]isradipine-labeled LTCCs as 54% of solubilized (+)-[H-3]isradipine-binding activity was specifically immunoprecipitated by alpha 1C antibodies. Phosphorylation of immunopurified alpha 1C with cAMP-dependent protein kinase revealed the existence of an additional 240-kDa species (long form), that remained undetected in Western blots. Fifty seven percent of labeled LTCCs were immunoprecipitated by an anti-beta-antibody directed against all known beta-subunits. Isoform-specific antibodies revealed that these mainly corresponded to beta 1b- and beta 3-subunits. We found beta 2- and beta 4-subunits to be major constituents of cardiac and brain L-type channels, respectively, but not part of L-type channels in RINm5F cells. We conclude that alpha 1C is a major constituent of dihydropyridine-labeled LTCCs in RINm5F cells, its long form sewing as a substrate for cAMP-dependent protein kinase. beta 1b- and beta 3-Subunits were also found to associate with L-type channels in these cells. These isoforms may therefore represent biochemical targets for the modulation of LTCC activity in RINm5F cells.