| Ca2+-triggered exocytosis plays a central role in synaptic transmission. Amperometry was used to investigate the factors affecting fusion and fusion pore kinetics, including temperature, membrane bending energy, and synaptotagmin-phosphatidylserine (syt-PS) interactions, in Ca2+-triggered excoytosis. Three main conclusions have been drawn from this work.;Highly temperature-dependent kinetic parameters include the frequency of exocytotic events and fusion pore lifetime, suggesting that these processes require structural rearrangements of proteins or lipids or both. The weak temperature dependence of spike shape parameters suggests that after the fusion pore has started to expand, diffusion is the only kinetically limiting step. This indicates that the content of a vesicle is expelled completely after fusion pore expansion.;The dependence of fusion pore stability on vesicle size leads to a nonexponential fusion pore lifetime distribution. Perturbing membrane curvature in opposite directions with lysophosphatidylcholine and oleic acid changed the size sensitivity of fusion pore lifetime in opposite ways. These effects are opposite to those observed in lipidic fusion pores. This was further supported by a theoretical model based on the elastic resistance of lipid bilayers to bending. Thus, during Ca2+-triggered exocytosis, membrane bending energy presents an obstacle to fusion pore dilation rather than to fusion pore formation. In contrast to viral fusion, Ca2+-triggered exocytosis begins with a proteinaceous or proteolipid fusion pore, with unstressed lipid bilayer, and evolves as it dilates to a lipidic pore, containing highly curved membrane.;Elevating PS in the plasma membrane increased Ca2+-triggered vesicle fusion and prolonged fusion pore openings. This was correlated with syt-PS binding ability. Overexpression of syt mutations and syt isoforms with distinct syt-PS binding ability altered fusion pore stability, further confirming that the formation of syt-PS complex drives fusion pore opening, and stabilize fusion pore by retarding the transition from open state to dilation state.;In conclusion, exocytosis is a higly temperature dependent event, which is controlled by membrane bending energy and syt-PS interactions. Multiple factors may coordinate to regulate release and fusion pore kinetics. |
