SNARE Copy Number Determines the Size and Kinetic Properties of Nascent Fusion Pores

Abstract

The first step in neurotransmitter and hormone release is the formation of a fusion pore. Capacitance measurements revealed that pore size can range from 0.5 to 2 nm in neuroendocrine cells. However, the mechanisms that determine pore size and dynamics are largely unknown due to the low abundance and ephemeral nature of these crucial structures. Here, we reduced the number of available SNAREs in neurons and observed changes in transmitter release suggestive of alterations in fusion pore properties. To address this possibility, we first employed reconstituted nanodisc/liposome-based fusion assays, using optical sensors to detect cargo flux through nascent pores. Ensemble and single vesicle measurements revealed that increasing the number of SNARE complexes enhances the rate of release of a given cargo, and enables the escape of larger cargos. To determine whether this was due to changes in pore size versus stability, we developed a novel reconstitution approach, based on nanodiscs and planar lipid bilayer electrophysiology, that affords µsec time resolution at the single event level. Remarkably, both parameters were affected by SNARE copy number. Increasing the total number of v-SNAREs per nanodisc from three to five caused a two-fold increase in pore size and decreased the rate of pore closure by two orders of magnitude. Moreover, trans-SNARE pairing was dynamic as perturbation of v-/t-SNARE zippering disrupted the stability of fusion pores, particularly at the base of the SNARE complex. In summary, trans-SNARE complexes are metastable and the number of SNAREs recruited to drive fusion dictates fundamental properties of individual pores.