Control of Ca2+ channel current and exocytosis in rat lactotrophs by basally active protein kinase C and calcineurin.
Modulation of voltage-activated Ca2+ channel activity by phosphorylation was studied in metabolically intact voltage-clamped rat lactotrophs. Experiments using Ba2+ as a charge carrier indicated that a phorbol ester protein kinase C activator stimulates high-voltage-activated Ca2+ channel currents, but has no effect on low-voltage-activated currents. Extracellular application of structurally and mechanistically distinct protein kinase C inhibitors (staurosporin, H7, calphostin C, chelerythrine and Ro 31-8220) preferentially inhibited the high-voltage-activated Ba2+ current. This suggests that protein kinase C is required for maintainance of Ca2+ channel activity even in the absence of modulators. Cyclosporin A, an inhibitor of the Ca2+/calmodulin-dependent protein phosphatase calcineurin, increased the high-voltage-activated Ca2+ channel current, and staurosporin reversed this effect. Thus, dephosphosphorylation by calcineurin may limit basal Ca2+ channel activity. Time-domain monitoring of cellular capacitance changes demonstrated that cyclosporin A and 12-O-tetradecanoyl-phorbol-13-acetate do not affect exocytosis at a hyperpolarized potential, but each enhances depolarization-induced exocytosis. Facilitation of exocytosis by cyclosporin A differed from 12-O-tetradecanoyl-phorbol-13-acetate in that it was biphasic. The delayed facilitation induced by cyclosporin A could be accounted for by stimulation of the voltage-gated Ca2+ current. These results suggest that the high-voltage activated Ca2+ channel current in rat lactotrophs is determined by the opposing basal activities of protein kinase C and calcineurin. Furthermore, it is concluded that the regulation of Ca2+ channels by protein kinase C and calcineurin affects depolarization-induced exocytosis.
Fomina
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Department of Pharmacology, University of Pittsburgh, PA 15261, U.S.A.
Levitan
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D000818:Animals; D001464:Barium / Q000378:metabolism; D019703:Calcineurin; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D002478:Cells, Cultured; D004558:Electric Stimulation; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000201:enzymology / Q000502:physiology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D015220:Calcium Channels; D002148:Calmodulin-Binding Proteins; D004791:Enzyme Inhibitors; D001464:Barium; D011493:Protein Kinase C; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases