Uncoupling the roles of synaptotagmin I as a dual Ca(2+) sensor during endo- and exocytosis of synaptic vesicles

Synaptotagmin I (syt1) is required for normal rates of synaptic vesicle endo- and exocytosis. However, whether the kinetic defects observed during endocytosis in syt1 knock-out neurons are secondary to defective exocytosis, or whether syt1 directly regulates the rate of vesicle retrieval, remains unresolved. In order to address this question, it is necessary to dissociate these two activities. Here, we have uncoupled the function of syt1 in exo- and endocytosis by re-targeting of the protein, or via mutagenesis of its tandem C2-domains; the impact of these manipulations on exo- and endocytosis were analyzed via electrophysiology, in conjunction with optical imaging of the vesicle cycle. These experiments uncovered a direct role for syt1 in endocytosis. Surprisingly, either C2-domain of syt1 - C2A or C2B - was able to function as Ca(2+)-sensor for endocytosis. Hence, syt1 functions as a dual Ca(2+) sensor for both endo- and exocytosis, potentially coupling these two limbs of the vesicle cycle.