The C2B domain of synaptotagmin is a Ca2+-sensing module essential for exocytosis

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis. Synaptotagmin spans the vesicle membran e once and possesses a large cytoplasmic domain that contains two C2 domain s, C2A and C2B. Multiple Ca2+ ions bind to the membrane proximal C2A domain . However, it is not known whether the C2B domain also functions as a Ca2+- sensing module. Here, we report that Ca2+ drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomeri zation of multiple isoforms of the protein. These effects of Ca2+ are media ted by a set of conserved acidic Ca2+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed t he function of oligomerization using a dominant negative approach. Two dist inct reagents that block synaptotagmin clustering potently inhibited secret ion from semi-intact PC12 cells. Together, these data indicate that the Ca2 +-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.