The Neuronal Priming Complex

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DESCRIPTION (provided by applicant): Although we have made great strides in understanding Ca+2-dependent exocytosis in the neuron, there is still not a clear understanding of the relationship between Ca+2 and exocytosis. An accurate view of this process is fundamental to understanding brain function, and crucial to the future treatment of neurological diseases. The N-type Ca+2 channel interacts with the exocytosis proteins syntaxin and SNAP-25; however, the structural basis of these interactions is still not clear. Through detailed primary and secondary structural analysis, we found that the N-type Ca+2 channel possesses a SNARE-like motif that may mediate its interactions with syntaxin and SNAP-25. Aim 1 of this R21 project will test the hypothesis that the N-type Ca+2 channel acts as a Q-SNARE, and that the interactions with syntaxin and SNAP-25 follow some of the same structural patterns observed in the cis-SNARE. This Ca+2 channel "SNARE" is not implicated in the fusogenic activities of the syntaxin-SNAP-25.synaptobrevin complex, but may serve to localize these components to the Ca+2 channel immediately before exocytosis. Taking advantage of the unique properties of the recombinant SNARE proteins, we expect to show that the synprint domain of the N-type Ca+2 channel interacts with syntaxin and SNAP-25 in a similar manner as do other SNARE proteins. Aim 2 will lay the foundation for further structural work on human synaptotagmin 1 C2A-C2B, the Ca+2 sensor in exocytosis. This protein also interacts with the Ca+2 channel, syntaxin and SNAP-25 in a multiprotein complex. As a prelude to eventual multiprotein analysis, we will refine the crystallization conditions of synaptotagmin using a variety of innovative crystallographic techniques designed to take advantage of synaptotagmin's unique lipid-associating properties. Once completed, these two aims will provide us with the tools and understanding to further define this protein's role in Ca+2-dependent exocytosis. Under future R01 funding we will be able to extend this work to more extensive biophysical and crystallographic studies of this unique SNARE complex, and complete the high-resolution crystal structure of human synaptotagmin 1 C2A-C2B.