DISTINCT CA2-PROPERTIES OF SYNAPTOTAGMINS - DEFINITION OF CANDIDATE CA2+ SENSORS FOR THE FAST AND SLOW COMPONENTS OF NEUROTRANSMITTER RELEASE( AND SR2+ BINDING)

Ca2+-dependent neurotransmitter release consists of at least two compo nents: a major fast component that is insensitive to Sr2+ and a minor slow component that is potentiated by Sr2+ (Goda, Y., and Stevens, C. F. (1994) Proc. Natl. Acad. U. S. A. 91, 12942-12946). These results s uggest that at least two Ca2+ sensors act in synaptic vesicle fusion w ith distinct Ca2+ and Sr2+ binding properties. We have now investigate d the relative Ca2+ and Sr2+ binding activities of synaptotagmins to e valuate their potential roles as Ca2+ sensors for the fast and slow co mponents. Our results demonstrate that the first C-2 domains of synapt otagmins I, II, III, V, and VII have very similar Ca2+ requirements fo r phospholipid binding (range of EC(50) = 2.6 mu M to 5.0 mu M), but d istinct Sr2+ requirements (EC(50) range = 23 mu M to 133 mu M); synapt otagmin I and II had the lowest Sr2+ affinity, and synaptotagmin III t he highest Sr2+ affinity. Purified synaptotagmin I from bovine brain e xhibited similar properties as its recombinant first C-2 domain, sugge sting that the first C-2 domain fully accounts for its Ca2+-dependent phospholipid binding properties. Sr2+ was unable to trigger syntaxin b inding by synaptotagmin I at all concentrations tested, whereas it was effective for synaptotagmin III. These results suggest that different C-2 domains have distinct Sr2+ binding properties. They support the h ypothesis that synaptotagmins localized on the same vesicle perform di stinct functions, with synaptotagmins I and II serving as candidate Ca 2+ sensors for the fast component in release and synaptotagmin III for the slow component.