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L-Type Calcium Channels in Insulin-Secreting Cells: Biochemical Characterization and Phosphorylation in RINm5F Cells

Pharmazeutisches Institut, Lehrstuhl Pharmakologie (H.S., U.K., A.B., M.R., H.P.T.A.) Universität Tübingen D-72076 Tübingen, Germany
Max Delbrück Centrum für Molekulare Medizin (H.H., I.M.) Cardiology D-13125 Berlin, Germany
Pfizer (M.K.A.) Central Research Division Groton, Connecticut 06340
Institut für Biochemische Pharmakologie (J.S., M.F., T.N.C.) Universität Innsbruck A-6020 Innsbruck, Austria

Opening of dihydropyridine-sensitive voltage-dependent L-type Ca²⁺-channels (LTCCs) represents the final common pathway for insulin secretion in pancreatic ß-cells and related cell lines. In insulin-secreting cells their exact subunit composition is unknown. We therefore investigated the subunit structure of (+)-[³H]isradipine-labeled LTCCs in insulin-secreting RINm5F cells. Using subunit-specific antibodies we demonstrate that 1C subunits (199 kDa, short form) contribute only a minor portion of the total 1 immunoreactivity in membranes and partially purified Ca²⁺-channel preparations. However, 1C forms a major constituent of (+)-[³H]isradipine-labeled LTCCs as 54% of solubilized (+)-[³H]isradipine-binding activity was specifically immunoprecipitated by 1C antibodies. Phosphorylation of immunopurified 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-ß-antibody directed against all known ß-subunits. Isoform-specific antibodies revealed that these mainly corresponded to ß1b- and ß3-subunits. We found ß2- and ß4-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 1C is a major constituent of dihydropyridine-labeled LTCCs in RINm5F cells, its long form serving as a substrate for cAMP-dependent protein kinase. ß1b- and ß3-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.

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