Fusion Between v-SNARE Nanodiscs and “Flipped” t-SNARE Cells: Control of Fusion Pore Nucleation and Lifetimes by SNARE Protein Transmembrane Domains

Abstract

A key step during neurotransmitter or hormone release (exocytosis) is the formation of a nanometer-sized fusion pore that connects the plasma membrane to the synaptic or secretory vesicle. The pore can flicker open and closed repeatedly before dilating or resealing irreversibly. Pore dynamics affect vesicle recycling, amount and kinetics of released cargo, and activation of downstream events. The fusion pore is clearly a key intermediate; however, factors regulating its dynamics are poorly understood, in large part due to a lack of biochemically defined single-pore assays.We have developed a novel assay that probes single fusion pores formed between ∼16 nm flat lipid bilayer nanodiscs (NDs) reconstituted with neuronal/exocytotic vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (v-SNARE) proteins and cells engineered to express cognate “flipped” target membrane t-SNAREs on their surfaces, with the SNARE domain facing the extracellular medium. Conductance through single, voltage-clamped fusion pores directly reports sub-millisecond pore dynamics. Pore currents fluctuated, briefly 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 putative interactions between v- and t-SNARE transmembrane domains (TMDs) promote, but are not essential for pore nucleation. Surprisingly, the same modifications that reduced pore nucleation rates dramatically prolonged pore lifetimes. Our results indicate that rod-shaped, post-fusion cis-SNARE complexes that poorly fit the highly curved fusion site rapidly vacate the pore region, leaving pore properties to be determined by lipids. In contrast, mutants deficient in TMD-zippering prevented pore resealing for >60 s, possibly due to their post-fusion Y-shape. Thus, post-fusion geometry of the proteins may determine pore stability, analogous to the well-known effects of lipid geometry on highly curved fusion intermediates.