Control of Fusion Pore Nucleation and Dynamics by SNARE Protein Transmembrane Domains

Abstract

Membrane fusion is a fundamental biological process, whose initial stages have been observed in hormone-secreting cells and neurons using electrophysiological and electrochemical methods. The initial 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 events such as vesicle recycling and release kinetics, but pore properties are poorly known, because fusion pores are transient, and biochemically defined assays with single-pore sensitivity are lacking. We isolated single flickering pores connecting v-SNARE-reconstituted nanodiscs to cells ectopically expressing cognate, “flipped” t-SNAREs, voltage-clamped in the cell-attached configuration. Currents through such pores directly report sub-millisecond single-pore dynamics. We found that interactions between v- and t-SNARE transmembrane domains (TMDs) observed in a recent crystal structure promote, but are not essential for pore nucleation. Surprisingly, TMD interactions also affected pore lifetimes. Rod-shaped, post-fusion cis-SNARE complexes vacated the highly curved fusion site where they fit poorly, leaving pore properties to be determined largely by lipid bilayers. In contrast, Y-shaped mutants deficient in TMD-zippering lingered at the fusion site, preventing pore resealing for >60 s. Thus, post-fusion geometry of the proteins determines pore stability, analogous to the well-known effects of lipid geometry on highly curved fusion intermediates.