Activity-Dependent Fusion Pore Dilation Mediated by a Dynamin I-Syndapin Pathway

Abstract

SNAP25 is a t-SNARE protein mediating exocytosis in neuronal cell types. Previous results with a construct lacking the nine C-terminal residues (SNAP25D9) showed changed fusion pore properties, suggesting a model for fusion pore mechanics that couple C-terminal zipping of the SNARE complex to the opening of a proteolipid fusion pore (Fang 2008 PNAS,105:15388). The deleted fragment contains the positively charged residues R198andK201, which are located in layers 7-8 of the SNARE complex at its C-terminal end. To study the role of these two residues in the structure and dynamics of the fusion pore, carbon fiber amperometry and cell-attached capacitance measurement of individual fusion and release events were performed using bovine chromaffin cells overexpressing wild type SNAP-25 or the point mutation constructs R198Q/E, K201Q/E to substitute the positively charged amino acid with a neutral or positively charged one. The R198E/K201E mutants increased fusion pore duration similar to SNAP25D9. Furthermore, the R198E/R198Q also showed a smaller fusion pore conductance. These results support the conclusion that fusion pore properties depend on the precise arrangements at the C-terminal end of the SNARE complex. Supported by NIH grant GM085808. 2207-Pos Board B193Activity-Dependent Fusion Pore Dilation Mediated by a Dynamin I-Syndapin Pathway Prattana Samasilp, Bryan Doreian, Shyue-An Chan, Corey Smith. Chormaffin cells of adrenal medulla serve a primary role in setting a proper homeostatic status under basal sympathetic tone as well as a physiological response to sympathetic stress. In order to function under these diverse conditions, chromaffin cells exhibit a differential release of catecholamine and co-packaged peptide transmitter molecules. Under basal sympathetic firing, catecholamine is selectively secreted through a restricted fusion pore of ''Kiss and Run'' exocytic mode. On the other hand, under acute stress, elevated sympathetic activity increases cytosolic calcium, leading to dilation of fusion pore and finally full granule collapse into plasma membrane. Both catecholamine and peptide transmitters are expelled in this condition. Thus, activity-dependent differential transmitter release is regulated by fusion pore dilation. Previous studies have shown that dynamin I plays a critical role in controlling fusion pore dilation in chromaffin cells. Here, we employ electrochemical, electrophysiological and fluorescence based approaches to investigate the molecular mechanism responsible for dynamin I-dependent fusion pore dilation. We show that syndapins (synaptic dynamin-associated proteins) are a primary molecular component of dynamin I-dependent fusion pore regulation. Disruption of dynamin I-syndapin interaction decreases normal activity-mediated catecholamine release. Our results suggest that fusion pore dilation is regulated by a dynamin I-syndapin-dependent signaling mechanism. 2208-Pos Board B194Oxidation and Palmitoylation of SNAP-25B In Vitro Nozomi Ogawa, Alex DaBell, Dixon J. Woodbury. SNAP-25 plays a cr...