title: An inhibitory role of calcineurin in endocytosis of synaptic vesicles at nerve terminals of Drosophila larvae.
abstract: In this study, we tested a hypothesis that activation of calcineurin, Ca2+/calmodulin-dependent protein phosphatase 2B, is an initiating signal for synaptic vesicle endocytosis. We examined effects of calcineurin inhibitors, cyclosporin A or FK506 and calmodulin inhibitors on stimulus-induced FM1-43 uptake into nerve terminals of Drosophila larvae. Fluorescent FM1-43 labels recycling synaptic vesicles in nerve terminals. Pretreatment with cyclosporin A (5-40 microM) or with FK506 (5-10 microM) enhanced FM1-43 uptake induced by high (60 mM) K+ in a dose-dependent manner. The effect required some preincubation time of about 10 min. The nerve terminals loaded with FM1-43 were destained by electrical nerve stimulation in the cyclosporin A-pretreated preparations, confirming that FM1-43 was taken up into synaptic vesicles. Pretreatment with rapamycin (2 or 20 microM), a structural analog of FK506 which has no effect on calcineurin, or calyculin A (0.3-50 nM), an inhibitor of protein phosphatase 1 and 2A, had no detectable effect on FM1-43 uptake. On the other hand, pretreatment with trifluoperazine (1-50 microM) or with phenoxybenzamine (100 microM), inhibitors of calmodulin, enhanced FM1-43 uptake. Since endocytosis is coupled with exocytosis, it is possible that the enhancement of FM1-43 uptake results from facilitation of exocytosis. However, the frequency of spontaneous junctional potentials and the mean amplitude of evoked potentials did not change after the cyclosporin A treatment, suggesting that the exocytosis process was not significantly affected by the drug. Furthermore, we can temporally separate synaptic vesicle exocytosis and endocytosis in a Drosophila mutant, shibire (shi(ts1)). By taking advantage of this mutation, we showed that cyclosporin A and trifluoperazine enhanced synaptic vesicle recycling by directly acting on the endocytotic process. Present results are not compatible with the hypothesis, but suggest that calcineurin inhibits synaptic vesicle recycling.
authors.affiliation: Institute for Behavioral Sciences, Gunma University School of Medicine, Maebashi, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Kuromi
authors.forename: M
authors.initials: M
authors.lastname: Yoshihara
authors.forename: Y
authors.initials: Y
authors.lastname: Kidokoro
mesh_terms: D000818:Animals; D019703:Calcineurin; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002148:Calmodulin-Binding Proteins / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004331:Drosophila melanogaster / Q000502:physiology*; D004558:Electric Stimulation; D004594:Electrophysiology; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007814:Larva; D008564:Membrane Potentials / Q000502:physiology; D018485:Muscle Fibers, Skeletal / Q000502:physiology; D009154:Mutation; D009411:Nerve Endings / Q000187:drug effects / Q000502:physiology*; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D011188:Potassium / Q000494:pharmacology; D054645:Protein Phosphatase 1; D013572:Synaptic Vesicles / Q000187:drug effects / Q000502:physiology*
chemical_list: D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D004791:Enzyme Inhibitors; D007166:Immunosuppressive Agents; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D011188:Potassium