Project Summary/Abstract Neurons and endocrine cells release signaling molecules by Ca2+‐triggered exocytosis. Ca2+ enters a nerve terminal or endocrine cell, binds to a Ca2+ sensor protein, and triggers the fusion of the vesicle membrane with the plasma membrane to expel some or all of the vesicle content into the extracellular space. To explore the mechanisms of exocytosis our research focuses on the fusion pore, the initial aqueous passage between the vesicle interior and the outside of a cell. All secreted molecules pass through a fusion pore, which is strategically situated to exert finely tuned control over secretion. By measuring amperometry, capacitance, and miniature postsynaptic currents, we probe fusion pores at the single‐pore level to track structural transitions and monitor responses to biological signals. Studies of the fusion pore have given us valuable insights into the roles of specific proteins in the control of membrane fusion. In the previous funding cycle we showed that the vesicle SNARE protein synaptobrevin alters transmitter flux through synaptic fusion pores in hippocampal neurons. In a remarkable parallel with our earlier work on endocrine fusion, the implicated synaptic fusion pore residues align precisely with those of endocrine fusion pores. We showed that another major vesicle protein, synaptophysin, influences exocytosis at multiple stages as fusion pores open and expand. Aim 1 will complete an ongoing effort to explore the role of the transmembrane domains of synaptophysin in the initial fusion pore. Turning from initial fusion pores to late‐stage fusion pore, we recently developed a new method for analyzing fusion pore dynamics during spikes in amperometric recordings from endocrine cells. This method tracks fusion pore permeability as vesicles lose catecholamine. This led to the novel findings that the pore sequentially expands, contracts, and settles into a metastable state. Aim 2 will use this method to investigate late‐stage fusion pores to address long‐standing questions about the biological control of vesicle content expulsion. We will probe late‐ stage fusion pores for control by lipid bilayer elasticity, Ca2+, hormone content, GPCR signaling, synaptotagmin, and synaptophysin/dynamin. Along a related front, we have developed better ways to study synaptic fusion pores. Co‐ cultures between neurons and HEK 293 cells provide a system for studying synaptic transmission with greatly enhanced resolution. Aim 3 will use this new co‐culture system to study synaptic release and determine how synaptic fusion pores are controlled by bilayer elasticity, Ca2+, synaptotagmin, and synaptophysin. This work will explore the largely unknown behavior of synaptic fusion pores and their dynamic control of synaptic release. These three aims will extend our understanding of initial fusion pores, and open up an exciting new line of investigation into how late‐stage fusion pores expand and contract in response to biological signal...