A low-affinity Ca 2+ /H + -antiport was described in the membrane of mammalian brain synaptic vesicles. Electrophysiological studies showed that this antiport contributes to the extreme brevity of excitation-release coupling in rapid synapses. Synaptotagmin-1, a vesicular protein interacting with membranes upon low-affinity Ca 2+ -binding, plays a major role in excitation-release coupling, by synchronizing calcium entry with fast neurotransmitter release. Here, we report that synaptotagmin-1 is necessary for expression of the vesicular Ca /H + -antiport activity is detectable only in acidic organelles expressing functional synaptotagmin-1. In contrast, synaptotagmin-1-deficient cells -and cells where transgenically encoded synaptotagmin-1 was acutely photo-inactivated -were devoid of any Ca 2+ /H + -antiport activity. Therefore, in addition to its previously described functions, synaptotagmin-1 is involved in a rapid vesicular Ca 2+ sequestration through a Ca It has long been known that synaptic vesicles and other secretory granules take up Ca 2+ by ATP-dependent mechanisms (Isra€ el et al. 1980;Michaelson et al. 1980;Parekh 2008). Two distinct mechanisms contribute to transport calcium into vesicles: a high-affinity Ca 2+ -ATPase pump, and a low-affinity Ca 2+ /H + -antiport, which was extensively investigated using synaptic vesicles isolated from mammalian brain cortex. The vesicular Ca 2+ /H + -antiport is activated only at high Ca 2+ concentrations (K 0.5 = 217 lM; maximum velocity at~500-600 lM). Its activity depends on the DpH across the vesicle membrane, and therefore on the vacuolar H + -transporting ATPase (V-ATPase) (Gonc ßalves et al. 1998(Gonc ßalves et al. , 1999b(Gonc ßalves et al. , 2000.It is expected that the high-affinity Ca 2+ -pumps transport Ca 2+ ions into vesicles (and into other organelles) when the synapse is in resting state, keeping the cytosolic Ca 2+ concentration ([Ca 2+ ] i ) at the very low physiological level. As for the Ca 2+ /H + -antiport, it would operate only at brief instants following a presynaptic action potential, when [Ca 2+ ] i abruptly raises to sub-millimolar levels in discrete spots (nano-domains) close to the inner mouth of voltage-operated calcium channels (VOCCs) (Llinas et al. 1992 Searching for a molecular counterpart of the vesicular Ca 2+ /H + -antiport activity, we suspected that synaptotagmins might be involved. Synaptotagmins form a family of proteins associated with synaptic vesicles. Synaptotagmin-1 (SYT-1) has a conserved N-terminal inside the vesicle and two C2 domains facing the cytosol, which are able to bind Ca 2+ with low affinity (60 lM to 600 lM) (Takamori et al. 2006;Chapman 2008). SYT-1 also binds in a dynamic and Ca 2+ -dependent manner to the hetero-trimeric protein complex SNARE (VAMP/synaptobrevin + SNAP-25+ syntaxin), to other proteins (complexin, etc.), to VOCCs and to phospholipids. SYT-1 and the above proteins form between vesicles and the plasma membrane a multimolecular complex, which is important for efficient excitation-...