Roles of G-proteins and cytoskeletal porteins in transmitter release.

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The aim of this research project is to clarify the roles of GTP-binding proteins and cytoskeletal proteins in the mechanism of transmitter release.Previously we have suggested that Go, one of heterotrimeric GTP-binding proteins, negatively regulates Ca^<2+>-dependent release of catecholamine in permeabilized chromaffin cells.We also have suggested a possible involvement of myosin light chain kinase in the mechanism of Ca^<2+>-dependent exocytosis.In the project, we attempted to elucidate the mechanism whereby Ca^<2+>-dependent release is regulated by Go, and by myosin light chain kinase in permeabilized chromaffin cells.We have obtained the following results.1.By use of mastoparan, a peptide known to activate directly GTP-binding proteins, and by use of anti-Go antibodies, we found that the negative regulation by Go is on the process of ATP-dependent priming of exocytosis.This inhibitory Go was suggested to be activated by GAP43, and activation of protein kinase C seemed to stimulate Ca^<2+>-dependent release by abolishing the action of GAP43 on Go.2.We found that myosin light chain kinase is essential for ATP-dependent priming of exocytosis, by use of specific inhibitors of myosin light chain kinase, wortmannin and SM-1.Histochemical studies suggested that the inhibition of myosin light chain kinase inhibits reorganization of cortical F-actin, and thus suggested an important role of cortical F-actin for priming of chromaffin vesicles to the exocytotic site.