Distinct Ca and Sr Binding Properties of Synaptotagmins DEFINITION OF CANDIDATE Ca SENSORS FOR THE FAST AND SLOW COMPONENTS OF NEUROTRANSMITTER RELEASE*

Ca-dependent neurotransmitter release consists of at least two components: a major fast component that is insensitive to Sr and a minor slow component that is potentiated by Sr (Goda, Y., and Stevens, C. F. (1994) Proc. Natl. Acad. U. S. A. 91, 12942–12946). These results suggest that at least two Ca sensors act in synaptic vesicle fusion with distinct Ca and Sr binding properties. We have now investigated the relative Ca and Sr binding activities of synaptotagmins to evaluate their potential roles as Ca sensors for the fast and slow components. Our results demonstrate that the first C2 domains of synaptotagmins I, II, III, V, and VII have very similar Ca requirements for phospholipid binding (range of EC50 5 2.6 mM to 5.0 mM), but distinct Sr 21 requirements (EC50 range 5 23 mM to 133 mM); synaptotagmins I and II had the lowest Sr affinity, and synaptotagmin III the highest Sr affinity. Purified synaptotagmin I from bovine brain exhibited similar properties as its recombinant first C2 domain, suggesting that the first C2 domain fully accounts for its Ca -dependent phospholipid binding properties. Sr was unable to trigger syntaxin binding by synaptotagmin I at all concentrations tested, whereas it was effective for synaptotagmin III. These results suggest that different C2 domains have distinct Sr binding properties. They support the hypothesis that synaptotagmins localized on the same vesicle perform distinct functions, with synaptotagmins I and II serving as candidate Ca sensors for the fast component in release and synaptotagmin III for the slow component.