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 mu M) or with FK506 (5-10 mu M) 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 mu M), 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 mu M) or with phenoxybenzamine (100 mu M), 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. (C) 1997 Elsevier Science Ireland Ltd.