Synaptotagmin function in dense core vesicle exocytosis studied in cracked pc12 cells

Ca²⁺-triggered dense-core vesicle exocytosis in PC12 cells does not require vesicular synaptotagmins 1 and 2, but may use plasma membrane synaptotagmins 3 and 7 as Ca²⁺ sensors. In support of this hypothesis, C₂ domains from the plasma membrane but not vesicular synaptotagmins inhibit PC12 cell exocytosis. Ca²⁺ induces binding of both plasma membrane and vesicular synaptotagmins to phospholipids and SNAREs (soluble N-ethylmaleimide-sensitive attachment protein receptors), although with distinct apparent Ca²⁺ affinities. Here we used gain-of-function C₂-domain mutants of synaptotagmin 1 and loss-of-function C₂-domain mutants of synaptotagmin 7 to examine how synaptotagmins function in dense-core vesicle exocytosis. Our data indicate that phospholipid- but not SNARE-binding by plasma membrane synaptotagmins is the primary determinant of Ca²⁺-triggered dense-core vesicle exocytosis. These results support a general lipid-based mechanism of action of synaptotagmins in exocytosis, with the specificity of various synaptotagmins for different types of fusion governed by their differential localizations and Ca²⁺ affinities.