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Subnanometer fusion pores in spontaneous exocytosis of peptidergic vesicles
Vardjan, Nina ...
Kiss-and-run exocytosis, consisting of reversible fusion between the vesicle membrane and the plasma membrane, is considered to lead to full fusion after stimulation of vesicles containing classical ...transmitters. However, whether this is also the case in the fusion of peytidergic vesicles is unknown. Previously, wehave observed that spontaneous neuropeptide discharge from a single vesicle is slower than stimulated release, because of the kinetic constraints of fusion pore opening. To explore whether slow spontaneous release also reflects a relatively narrow fusion pore, we analyzed the permeation of FM 4-64 dye and HEPFS molecules through spontaneously forming fusion pores in lactotroph vesicles expressing synaptop Hluorin, a pH-dependent fiuorescent fusion marker. Confocal imaging showed that half of the spontaneous exocytotic events exhibited fusion pore openings associated with a change in synaptop Hluorin fluorescence but were impermeable to FM 4-64and HEPES. Together with membrane capacitance measurements, these findingsindicate an open fusion pore diameter <0.5 nm, much smaller than the neuropeptides. In stimulated cells, >70% of exocytotic events exhibited a larger, FM 4-64-permeable pore (>1 nm). Interestingly, capacitance measurements showed that the majority of exorytotic events in spontaneous and stimulated conditions were transient. Stimulation increased the frequency of transient events and the fusion pore dwell time but decreased the fraction of events with lowest measurable fusion pore. Kiss-and-run is the predominant mode of exocytosis in resting and in stimulated peptidergic vesicles. Stimulation prolongs the effective opening of the fusion pore and expands its primary subnanometer diameter to enable hormone secretion without full fusion.
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