Abstract
The initial, nanometer-sized connection between the plasma membrane and a hormone- or neurotransmitter-filled vesicle –the fusion pore– can flicker open and closed repeatedly before dilating or resealing irreversibly. Pore dynamics determine release and vesicle recycling kinetics, but pore properties are poorly known because biochemically defined single-pore assays are lacking. We isolated single flickering pores connecting v-SNARE-reconstituted nanodiscs to cells ectopically expressing cognate, “flipped” t-SNAREs. Conductance through single, voltage-clamped fusion pores directly reported sub-millisecond pore dynamics. Pore currents fluctuated, transiently returned to baseline multiple times, and disappeared ~6 s after initial opening, as if the fusion pore fluctuated in size, flickered, and resealed. We found that interactions between v- and t-SNARE transmembrane domains (TMDs) promote, but are not essential for pore nucleation. Surprisingly, TMD modifications designed to disrupt v- and t-SNARE TMD zippering prolonged pore lifetimes dramatically. We propose that the post-fusion geometry of the proteins contribute to pore stability.
Highlights
About fusion pore dynamics that can be extracted, is currently limited to a time-averaged pore openness[22]
Using the new single fusion pore assay, we show that interactions between v- and t-sensitive factor attachment protein receptors (SNARE) transmembrane domains (TMDs) promote, but are not essential for pore nucleation
We filled the tip of the pipette with ~1 μl buffer, and overlaid on top a solution containing 16 ± 2 nm diameter bilayer discs stabilized by the membrane scaffold protein (MSP)[20], each bearing 7–9 copies of the v-SNARE VAMP220
Summary
About fusion pore dynamics that can be extracted, is currently limited to a time-averaged pore openness[22]. Complex formation between the neuronal/exocytotic v-SNARE vesicle-associated membrane protein 2 (VAMP2, known as synaptobrevin-2) and the t-SNAREs syntaxin-1 (Stx1) and synaptosomal-associated protein 25 (SNAP25) starts from the membrane distal N-termini, proceeding in stages[24] toward the membrane proximal regions, resulting in a four-helix bundle (SNAREpin) that brings bilayers into close proximity. It is not known how a pore nucleates at this stage. Disrupting the putative v- and t-SNARE TMD interactions dramatically prolonged pore lifetimes
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