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Abstract

One of the central problems in neuroscience is to define the roles of presynaptic proteins in neurotransmitter release. Peptides from the binding sites of these proteins serve as valuable probes of protein function. Important criteria for such peptides include their ability to specifically perturb a defined protein-protein interaction and to be sufficiently soluble to permit microinjection. A number of strategies have been employed to design effective peptides. Successful strategies include making educated guesses based on protein primary sequence, using binding studies to define sites of protein-protein interaction, and designing peptides based on the three-dimensional structure of known sites of interaction. Once peptides have been synthesized, it is important to characterize their actions in vitro before introducing them into cells. Particularly valuable in this regard are affinity chromatography, to define the binding partners of the peptides, and measurements of the actions of peptides upon binary interactions between presynaptic proteins. Finally, a synapse suited to microinjection, such as the squid giant synapse, can be used to evaluate the functional and structural actions of the peptides. This chapter describes our recent work using peptides to elucidate pre-fusion roles of N-ethylmaleimide-sensitive factor (NSF), α-soluble NSF-attachment protein (α-SNAP), and complexin in regulating SNAP receptor (SNARE)-dependent membrane fusion at this synapse.