Genetic analysis of synaptotagmin-7 function in synaptic vesicle exocytosis

Synaptotagmin-7 is a candidate Ca2+ sensor for exocytosis that is at least partly localized to synapses. Similar to synaptotagmin-1, which functions as a Ca2+ sensor for fast synaptic vesicle (SV) exocytosis, synaptotagmin-7 contains C(2)A and C2B domains that exhibit Ca2+- dependent phospholipid binding. However, synaptotagmin-7 cannot replace synaptotagmin-1 as a Ca2+ sensor for fast SV exocytosis, raising questions about the physiological significance of its Ca2+-binding properties. Here, we examine how synaptotagmin-7 binds Ca2+ and test whether this Ca2+ binding regulates Ca2+-triggered SV exocytosis. We show that the synaptotagmin-7 C(2)A domain exhibits a Ca2+-binding mode similar to that of the synaptotagmin-1 C2A domain, suggesting that the synaptotagmin-1 and -7 C-2 domains generally employ comparable Ca2+-binding mechanisms. We then generated mutant mice that lack synaptotagmin-7 or contain point mutations inactivating Ca2+ binding either to both C-2 domains of synaptotagmin-7 or only to its C2B domain. Synaptotagmin-7-mutant mice were viable and fertile. Inactivation of Ca2+ binding to both C-2 domains caused an approximate to 70% reduction in synaptotagmin-7 levels, whereas inactivation of Ca2+ binding to only the C2B domain did not alter synaptotagmin-7 levels. The synaptotagmin-7 deletion did not change fast synchronous release, slow asynchronous release, or short-term synaptic plasticity of release of neurotransmitters. Thus, our results show that Ca2+ binding to the synaptotagmin-7 C-2 domains is physiologically important for stabilizing synaptotagmin-7, but that Ca2+ binding by synaptotagmin-7 likely does not regulate SV exocytosis, consistent with a role for synaptotagmin-7 in other forms of Ca2+-dependent synaptic exocytosis.