The C2B domain of synaptotagmin is a Ca(2+)-sensing module essential for exocytosis.
The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca(2+) 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 Ca(2+) ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca(2+)-sensing module. Here, we report that Ca(2+) 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 Ca(2)+ are mediated by a set of conserved acidic Ca(2)+ 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 Ca(2)+-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.
Desai
R C
RC
Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706, USA.
Vyas
B
B
Earles
C A
CA
Littleton
J T
JT
Kowalchyck
J A
JA
Martin
T F
TF
Chapman
E R
ER
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000502:physiology*; D020013:Calcium Signaling / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000502:physiology; D003594:Cytoplasmic Granules / Q000502:physiology; D004705:Endocytosis; D005089:Exocytosis / Q000502:physiology*; D007425:Intracellular Membranes / Q000502:physiology; D046911:Macromolecular Substances; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000502:physiology*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000502:physiology*; D016716:PC12 Cells; D020033:Protein Isoforms / Q000737:chemistry / Q000502:physiology; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D050863:Synaptotagmin I; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D050857:Synaptotagmins; D002118:Calcium