An inhibitory role of calcineurin in endocytosis of synaptic vesicles at nerve terminals of Drosophila larvae.

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.