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Published online 4 September 2000. doi:10.1083/jcb.150.5.1125
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© The Rockefeller University Press, 0021-9525/2000//1125 $5.00
The Journal of Cell Biology, Volume 150, Number 5, , 2000 1125-1136

Original Article

The C2b Domain of Synaptotagmin Is a Ca2+–Sensing Module Essential for Exocytosis



Radhika C. Desaia, Bimal Vyasa, Cynthia A. Earlesa, J. Troy Littletonb, Judith A. Kowalchyckc, Thomas F.J. Martinc, and E.R. Chapmana

a Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706
b Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin 53706
c Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706
Department of Physiology, SMI 129, University of Wisconsin, 1300 University Avenue, Madison, WI 53706.(608) 265-5512

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

© 2000 The Rockefeller University Press

J.T. Littleton's current address is Center for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA 02139.

Abbreviations used in this paper: SNARE, N-ethyl maleimide-sensitive factor attachment protein receptor; t-SNARE, target SNARE.


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