Dynamics and number of trans-SNARE complexes determine nascent fusion pore properties

Analysis at high temporal and spatial resolution shows that the number and dynamics of SNARE proteins available during exocytosis determines the size and stability of fusion pores. Membrane pores ruled by SNAREs A critical early event during exocytosis is the transient formation of fusion pores in the cell membrane. Edwin Chapman and colleagues provide insights into the regulation of the size and kinetics of these structures. They were intrigued that, in neurons, reducing the number of SNARE proteins, which mediate membrane fusion and thus pore formation, results in changes in both neurotransmitter release and fusion pores. To investigate how these alterations come about, they used in vitro reconstitution analyses with microsecond time resolution and single-event space resolution. The analyses reveal that the number of SNARE proteins affects not only the rate of cargo release from individual pores, but also the size and stability of the pores and, consequently, the size of the released cargo. This approach also reveals that mimicking the action of botulinum neurotoxin A reduces fusion pore stability. The fusion pore is the first crucial intermediate formed during exocytosis, yet little is known about the mechanisms that determine the size and kinetic properties of these transient structures 1 . Here, we reduced the number of available SNAREs (proteins that mediate vesicle fusion) in neurons and observed changes in transmitter release that are suggestive of alterations in fusion pores. To investigate these changes, we employed reconstituted fusion assays using nanodiscs to trap pores in their initial open state. Optical measurements revealed that increasing the number of SNARE complexes enhanced the rate of release from single pores and enabled the escape of larger cargoes. To determine whether this effect was due to changes in nascent pore size or to changes in stability, we developed an approach that uses nanodiscs and planar lipid bilayer electrophysiology to afford microsecond resolution at the single event level. Both pore size and stability were affected by SNARE copy number. Increasing the number of vesicle (v)-SNAREs per nanodisc from three to five caused a twofold increase in pore size and decreased the rate of pore closure by more than three orders of magnitude. Moreover, pairing of v-SNAREs and target (t)-SNAREs to form trans -SNARE complexes was highly dynamic: flickering nascent pores closed upon addition of a v-SNARE fragment, revealing that t