An antibody to synaptotagmin I facilitates synaptic transmission Shao-Ying Hua, 1 Merilee A. Teylan 1, * and Aylin Cimenser 2 1 Department of Biological Sciences, Barnard College, Columbia University, 3009 Broadway, New York, NY, 10027, USA 2 Center for Theoretical Neuroscience, Center for Neurobiology and Behavior, Kolb Research Annex, Columbia University, 1051 Riverside Drive, New York, NY 10032-2695, USA Keywords: EPSP rise time, neurotransmitter release, synaptic delay, synaptotagmin Abstract Proper functioning of the nervous system requires precise control of neurotransmitter release. Synaptotagmin, a synaptic vesicle protein, is crucial for the temporal control of neurotransmitter release. The mechanism of synaptotagmin function is still under debate. To investigate the mechanism by which synaptotagmin controls neurotransmitter release, we injected an antibody of rat synaptotagmin I into a crayfish motor axon. We found that the antibody enhanced synaptic transmission at crayfish neuromuscular junctions by increasing the amplitude of the evoked synaptic response. This effect was antibody-dose dependent. The antibody also reduced the rise time of the synaptic potentials. These effects were accompanied by a reduction in the Hill coefficient for Ca 2+ - dependence of synaptic transmission. Our findings support the hypothesis that synaptotagmin inhibits neurotransmitter release in the absence of Ca 2+ . Introduction Neurotransmitter release at synapses requires fusion of the synaptic vesicle membrane with the nerve terminal membrane. This fusion is precisely controlled by Ca 2+ through molecular interactions; the details of these interactions are still under investigation (Arac et al., 2006; Bhalla et al., 2006; Giraudo et al., 2006; Tang et al., 2006). Membrane fusion requires close proximity of the membranes, which can be achieved by tight binding of SNARE (soluble N-ethylmalei- mide-sensitive factor attachment protein receptor) proteins from the opposite membranes (Sollner et al., 1993). With all the required pre- and postsynaptic molecular reactions, synaptic transmission is yet several orders faster than most of the cellular reactions, which typically take minutes; while the delay of fast synaptic transmission is less than 1 ms (Katz & Miledi, 1965; Llina ´s et al., 1981; Sabatini & Regehr, 1996). In an effort to explain the highly efficient temporal control of the membrane fusion in neurotransmitter release, a model of molecular arrangement for fusion was proposed in a previous study, in which SNARE proteins form a partial complex at the release site before Ca 2+ influx (Hua & Charlton, 1999). The engagement of SNARE proteins before Ca 2+ entry ensures quick release of neuro- transmitters after Ca 2+ influx. However, there are two issues not explained by the partial SNARE complex model. To begin with, SNARE proteins interact with a very high binding affinity. This raises the question of how the SNARE protein interaction stops at a partial binding state. Secondly, the model does not explain how vesicle fusion is controlled by Ca 2+ influx. In search of the Ca 2+ sensor in synaptic transmission, synaptotag- min I was found to be able to couple Ca 2+ influx to SNARE-mediated membrane fusion (reviewed by Chapman, 2002). Synaptotagmin I has two Ca 2+ -binding motifs (C2A and C2B domains) in its cytoplasmic region. Upon binding with Ca 2+ , the C2 motifs partially penetrate into the lipid bilayer membranes with very rapid kinetics (Davis et al., 1999). The C2A domain also interacts with the SNARE complex (Chapman, 2002). These features make synaptotagmin suitable for a role in the temporal control of synaptic vesicle fusion. Two major hypotheses have been proposed to explain the role of synaptotagmin in Ca 2+ -induced vesicle fusion. Deletion of synapto- tagmin I has been shown to uncouple Ca 2+ influx and neurotrans- mitter release, which may simply suggest that synaptotagmin I stimulates membrane fusion upon Ca 2+ binding (DiAntonio & Schwarz, 1994; Geppert et al., 1994). In support of this hypothesis, Tucker et al. (2004) showed that in the presence of Ca 2+ , the cytoplasmic region of synaptotagmin stimulated the SNARE-cata- lysed membrane fusion in a reconstituted system. An alternative hypothesis is based on the observations that suppression or reduction of the expression level of synaptotagmin leads to an increase in the rate of spontaneous neurotransmitter release despite a nearly complete elimination of the evoked release (Shoji-Kasai et al., 1992; DiAntonio et al., 1993; Littleton et al., 1993, 1994; Broadie et al., 1994; DiAntonio & Schwarz, 1994; Pang et al., 2006). These observations pointed to an inhibitory role of synaptotagmin in membrane fusion (reviewed by Popov & Poo, 1993). With the genetic approaches used in these studies, the synaptotagmin expression was greatly reduced or totally suppressed. In the present study, we used an antibody of rat synaptotagmin I to moderately reduce the function of synaptotagmin. Our results suggest that synaptotagmin controls neurotransmitter release by blocking vesicle fusion in the absence of Ca 2+ . Correspondence: Dr S.-Y. Hua, as above. E-mail: shua@barnard.edu *Present address: Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. Received 23 December 2006, revised 31 March 2007, accepted 20 April 2007 European Journal of Neuroscience, Vol. 25, pp. 3217–3225, 2007 doi:10.1111/j.1460-9568.2007.05602.x ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd