Calcium sensing by the C2B domain of synaptotagmin

University Of Wisconsin-Madison, Madison WI

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Abstract

DESCRIPTION (provided by applicant): Precise signaling between neurons is required for normal brain function, and aberrant synaptic transmission results in nervous system disorders. Synaptic transmission requires calcium influx and the transduction of this calcium signal into release of neurotransmitter. The C2B domain of synaptotagmin is thought to act as the calcium sensor during exocytosis. Different C2B calcium binding mutants exhibit differences in effector interactions allowing a dissection of the role of these sites and interactions in distinct steps of fusion pore kinetics. The goal of this proposal is to test the hypothesis that C2B calcium binding mutants deficient in specific effector interactions show deficiencies in distinct kinetic steps of exocytosis during the opening and dilation of effusion pores and in synaptic transmission. These hypotheses will be tested by 1) assaying the ability of synaptotagmin C2B calcium binding mutants to bind presynaptic proteins in vitro, 2) performing amperometry on PC12 cells expressing these mutants to assess whether mutants deficient in effector interactions are deficient in the formation or dilation of fusion pores, and 3) determining whether these mutants are deficient in synaptic transmission in neurons.