Cholesterol Promotes Opening Of The Snare-Mediated Fusion Pore

SNARE proteins feature in almost all known intracellular membrane fusion events, such as exocytosis, the fusion of hormone or neurotransmitter filled vesicles with the plasma membrane to release their cargo. SNAREs have been shown to be the minimal membrane fusion machinery in vitro, driving fusion on timescales from 10-100 ms. A fundamental characteristic of the initial fusion pore is its flickering between open and closed states, with live cell electrochemical and electrophysiological measurements showing flickering timescales of ∼0.1 ms to 1 s during exocytosis. The flickering pore characteristics vary greatly, regulating the amount and the size of the released cargo. The molecular basis of this physiological regulation is not known. Here, we study flickering statistics of neuronal SNARE-mediated fusion pores in vitro for the first time. We used total internal reflection fluorescence microscopy (TIRFM) to measure fusion pores between reconstituted v-SNARE small unilamellar vesicles (SUVs) and cognate t-SNARE PEG-supported bilayers (SBLs). From transfer rates of fluorescently labeled vesicle lipids to the SBL, we measured the fraction of the time for which fusion pores are open on a single event basis. The accuracy was enhanced by single lipid fluorescence intensity, diffusivity and bleaching measurements. We find that increasing cholesterol content increases the openness of the fusion pore, the fraction of the time for which the pore connects the vesicle and SBL membranes (∼7-fold increase compared to ∼10% for cholesterol free bilayers). Cholesterol also strongly accelerated fusion pore initiation following vesicle docking. With increasing cholesterol concentration, SNAREs open the fusion pore a greater fraction of the time than when SNAREs are absent (∼2-3-fold greater with SNAREs than without). Other lipid components tested (PC, PS, PE) had a minimal effect on pore openness. Thus, we find that cholesterol promotes fusion pore opening in SNARE-mediated fusion.