SUMMARY Synaptic transmission involves a fast synchronous phase and a slower asynchronous phase of neurotransmitter release that are regulated by distinct Ca2+ sensors. While the Ca2+ sensor for rapid exocytosis, synaptotagmin I, has been studied in depth, the sensor for asynchronous release remains unknown. In a screen for neuronal Ca2+ sensors that respond to changes in [Ca2+] with markedly slower kinetics than synaptotagmin I, we observed that Doc2, another Ca2+, SNARE, and lipid binding protein, operates on time scales consistent with asynchronous release. Moreover, up- and down-regulation of Doc2 expression levels in hippocampal neurons increased or decreased, respectively, the slow phase of synaptic transmission. Synchronous release, when triggered by single action potentials, was unaffected by manipulation of Doc2, but was enhanced during repetitive stimulation in Doc2 knockdown neurons potentially due to greater vesicle availability. In summary, we propose that Doc2 is a Ca2+ sensor that is kinetically tuned to regulate asynchronous neurotransmitter release.

SUMMARY Synaptotagmin-I (syt) is a Ca2+ sensor that triggers synchronous neurotransmitter release. The first documented biochemical property of syt was its ability to aggregate membranes in response to Ca2+. However, the mechanism and function of syt-mediated membrane aggregation are poorly understood. Here, we demonstrate that syt-mediated vesicle aggregation is driven by trans interactions between syt molecules bound to different membranes. We observed a strong correlation between the ability of Ca2+-syt to aggregate vesicles and to stimulate SNARE-mediated membrane fusion. Moreover, artificial aggregation of membranes - using non-syt proteins - also efficiently promoted fusion of SNARE-bearing liposomes. Finally, using a modified fusion assay, we observed that syt drives the assembly of otherwise non-fusogenic individual t-SNARE proteins into fusion competent heterodimers, in an aggregation-independent manner. Thus, membrane aggregation and t-SNARE assembly appear to be two key aspects of Ca2+-syt-regulated, SNARE-catalyzed fusion reactions.

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 Ca2+-sensor for endocytosis. Hence, syt1 functions as a dual Ca2+ sensor for both endo- and exocytosis, potentially coupling these two limbs of the vesicle cycle.