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Synaptotagmin 1 (Syt 1) functions as an essential [Ca.sup.2+] sensor for the fast but not slow component of [Ca.sup.2+]-triggered exocytosis. One hypothesis to account for this selective function, based on the close homology of Syt 1 with synaptotagmin 9 (Syt 9), is that these Syts are redundant for the slow but not the fast component of release. We now show, however, that Syt 9 has unique properties that set it apart from Syt 1. Different from Syt 1, endogenous Syt 9 does not associate [Ca.sup.2+] dependently or independently with soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) protein complexes, and the Syt 9 [C.sub.2]B domain does not form [Ca.sup.2+]/phospholipid complexes, whereas such complexes are essential for Syt 1 function. Nevertheless, the [C.sub.2]A domain of Syt 9 functions as a [Ca.sup.2+]-binding module, suggesting that Syts 1 and 9 are [Ca.sup.2+] sensors with similar [Ca.sup.2+]-binding sequences but distinct properties that indicate nonoverlapping functions. [C.sub.2] domain | exocytosis | neurotransmitter release | synaptic plasticity | membrane fusion

Inositol hexakisphosphate (InsP(6)) levels rise and fall with neuronal excitation and silence, respectively, in the hippocampus, suggesting potential signaling functions of this inositol polyphosphate in hippocampal neurons. We now demonstrate that intracellular application of InsP(6) caused a concentration-dependent inhibition of autaptic excitatory postsynaptic currents (EPSCs) in cultured hippocampal neurons. The treatment did not alter the size and replenishment rate of the readily releasable pool in autaptic neurons. Intracellular exposure to InsP(6) did not affect spontaneous EPSCs or excitatory amino acid-activated currents in neurons lacking autapses. The InsP(6)-induced inhibition of autaptic EPSCs was effectively abolished by coapplication of an antibody to synaptotagmin-1 C2B domain. Importantly, preabsorption of the antibody with a GST-WT synaptotagmin-1 C2B domain fragment but not with a GST-mutant synaptotagmin-1 C2B domain fragment that poorly reacted with the antibody impaired the activity of the antibody on the InsP(6)-induced inhibition of autaptic EPSCs. Furthermore, K(+) depolarization significantly elevated endogenous levels of InsP(6) and occluded the inhibition of autaptic EPSCs by exogenous InsP(6). These data reveal that InsP(6) suppresses excitatory neurotransmission via inhibition of the presynaptic synaptotagmin-1 C2B domain-mediated fusion via an interaction with the synaptotagmin Ca(2+)-binding sites rather than via interference with presynaptic Ca(2+) levels, synaptic vesicle trafficking, or inactivation of postsynaptic ionotropic glutamate receptors. Therefore, elevated InsP(6) in activated neurons serves as a unique negative feedback signal to control hippocampal excitatory neurotransmission.

Neurotransmitter release is triggered by cooperative Ca2+-binding to the Ca2+-sensor protein synaptotagmin-1. Synaptotagmin-1 contains two C2 domains, referred to as the C2A and C2B domains, that bind Ca2+ with similar properties and affinities. However, Ca2+ binding to the C2A domain is not required for release, whereas Ca2+ binding to the C2B domain is essential for release. We now demonstrate that despite its expendability, Ca2+-binding to the C2A domain significantly contributes to the overall triggering of neurotransmitter release, and determines its Ca2+ cooperativity. Biochemically, Ca2+ induces more tight binding of the isolated C2A domain than of the isolated C2B domain to standard liposomes composed of phosphatidylcholine and phosphatidylserine. However, here we show that surprisingly, the opposite holds true when the double C2A/B-domain fragment of synaptotagmin-1 is used instead of isolated C2 domains, and when liposomes containing a physiological lipid composition are used. Under these conditions, Ca2+ binding to the C2B domain but not the C2A domain becomes the primary determinant of phospholipid binding. Thus, the unique requirement for Ca2+ binding to the C2B domain for synaptotagmin-1 in Ca2+-triggered neurotransmitter release may be accounted for, at least in part, by the unusual phospholipid-binding properties of its double C2A/B-domain fragment.