Original Article

The C2B Domain of Synaptotagmin Is a Ca²⁺–sensing Module Essential for Exocytosis

Correspondence to: E.R. Chapman, Department of Physiology, SMI 129, University of Wisconsin, 1300 University Avenue, Madison, WI 53706. Fax:(608) 265-5512 E-mail:chapman{at}physiology.wisc.edu.

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca²⁺ sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, 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-oligomerization of multiple isoforms of the protein. These effects of Ca²+ are mediated by a set of conserved acidic Ca²+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion 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.

Key Words: oligomerization, membrane fusion, synprint, C2 domain, Ca²⁺ binding

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