The calcineurin-binding protein cain is a negative regulator of synaptic vesicle endocytosis

During neurotransmitter release, exocytosed neurotransmitter vesicles are r ecycled by endocytosis, which involves the assembly of a complex of endocyt ic proteins. Assembly of endocytic proteins into a functional complex depen ds on their dephosphorylation by calcineurin, a calcium-sensitive protein p hosphatase and the inhibitory target of immunosuppressive drugs cyclosporin A and FK506. Cain is a recently identified protein inhibitor of calcineuri n. We now provide evidence that cain is a component of the endocytic protei n complex. The proline-rich region of cain forms a stable association with the SH3 domain of amphiphysin 1. Using a transferrin uptake assay, we found that overexpression of cain in HEK293 cells blocks endocytosis as potently as expression of a dominant negative dynamin 1 construct. The use of other calcineurin inhibitors such as cyclosporin A and FK506 also blocks endocyt osis. Since binding of cain to amphiphysin 1 does not affect amphiphysin's interaction with other endocytic proteins, our results suggest that cain ne gatively regulates synaptic vesicle endocytosis by inhibiting calcineurin a ctivity, rather than sterically interfering with the assembly of the endocy tic protein complex.