"How to isolate and study the ‘fusion pore’, a key intermediate during exocytotic membrane fusion" Synaptic transmission underlies neuronal communication and hormones control many physiologic processes. Neurotransmitters and hormones are released via calcium-triggered exocytosis wherein cargo-filled vesicles fuse with the plasma membrane to release their contents to the extracellular space. Both hormones and neurotransmitters are released through fluctuating, nm-sized exocytotic fusion pores that can flicker open and shut multiple times. Cargo release and vesicle recycling depend on the fate of the pore, which may reseal or dilate irreversibly. Pore nucleation requires zippering between vesicle-associated v-SNAREs and target membrane t-SNAREs, but the mechanisms governing pore dynamics are not understood. We have developed methods to probe properties of single fusion pores, using both electrical and fluorescence methods, in biochemically defined settings. We isolated pores connecting v-SNARE-reconstituted nanodiscs to cells ectopically expressing cognate, “flipped” t-SNAREs. Conductance through single, voltage-clamped fusion pores directly report sub-millisecond pore dynamics. I will discuss our ongoing efforts to characterize mechanisms governing pore dynamics. These include subtle modifications of the transmembrane domains of SNAREs, molecular crowding at the pore’s waist, and lipid- and SNARE-binding of the calcium sensor for rapid exocytosis, Sytnaptotagmin-1.