Distinct Ca2+-dependent properties of the first and second C-2-domains of synaptotagmin I

Synaptotagmin I (SytI) is a synaptic vesicle protein that binds Ca2+ and is essential for fast, Ca2+-dependent neurotransmitter release in the hippocampus, suggesting that it serves as a Ca2+ sensor for exocytosis. Although SytI has two cytoplasmic C-2-domains, only the first C-2-domain was shown to exhibit Ca2+ regulation;it binds phospholipids and syntaxin in a Ca2+-dependent manner. By contrast, the second C-2-domain is inactive in these assays and only binds putative interacting molecules in a Ca2+-independent manner. We have now discovered in a yeast two-hybrid screen for SytI-interacting molecules that the C-2-domains of SytI interact with themselves. Using immobilized recombinant C-2-domains from SytI and SytII, we found that only the second but not the first C-2-domains of these synaptotagmins are capable of affinity-purifying native rat brain SytI and that this binding is Ca2+-dependent, suggesting that only the second C-2-domain is capable of a Ca2+ triggered self-association. A relatively high Ca2+ concentration (>100 mu m) is required for binding in the presence of Mg2+; Sr2+ and Ba2+ but not Mg2+ can substitute for Ca2+. Our data suggest that the second C-2-domain of SytI is also a Ca2+-regulated domain similar to the first C-2-domain but with distinct binding activities.