CALCIUM-DEPENDENCE OF NEUROTRANSMITTER RELEASE AND RATE OF SPONTANEOUS VESICLE FUSIONS ARE ALTERED IN DROSOPHILA SYNAPTOTAGMIN MUTANTS

Since the demonstration that Ca2+ influx into the presynaptic terminal is essential for neurotransmitter release, there has been much specul ation about the Ca2+ receptor responsible for initiating exocytosis. N umerous experiments have shown that the protein, or protein complex, b inds multiple Ca2+ ions, resides near the site of Ca2+ influx, and has a relatively low affinity for Ca2+. Synaptotagmin is an integral memb rane protein of synaptic vesicles that contains two copies of a domain known to be involved in Ca2+-dependent membrane interactions. Synapto tagmin has been shown to bind Ca2+ in vitro with a relatively low affi nity. In addition, synaptotagmin has been shown to bind indirectly to Ca2+ channels, positioning the protein close to the site of Ca2+ influ x. Recently, a negative regulatory role for synaptotagmin has been pro posed, in which it functions as a clamp to prevent fusion of synaptic vesicles with the presynaptic membrane. Release of the clamp would all ow exocytosis. Here we present genetic and electrophysiological eviden ce that synaptotagmin forms a multimeric complex that can function as a clamp in vivo. However, upon nerve stimulation and Ca2+ influx, all synaptotagmin mutations dramatically decrease the ability of Ca2+ to p romote release, suggesting that synaptotagmin probably plays a key rol e in activation of synaptic vesicle fusion. This activity cannot simpl y be attributed to the removal of a barrier to secretion, as we can el ectrophysiologically separate the increase in rate of spontaneous vesi cle fusion from the decrease in evoked response. We also find that som e syt mutations, including those that lack the second Ca2+-binding dom ain, decrease the fourth-order dependence of release on Ca2+ by approx imately half, consistent with the hypothesis that a synaptotagmin comp lex functions as a Ca2+ receptor for initiating exocytosis.