Direct Detection of Reconstituted, Snare-Mediated Fusion Pore Dynamics

Abstract

Exocytosis underlies release of neurotransmitters and hormones. Electrophysiological and electrochemical measurements from live cells have shown that the initial fusion pore is small (∼1 nm diameter) and can repeatedly flicker before either dilating fully, or closing permanently. The fraction of flickering pores and flicker characteristics vary with stimulation strength, regulating the amount and size of released cargo. The molecular mechanisms regulating fusion pore dynamics are not well understood, partly because in vitro techniques with sufficient resolution have been lacking. Here we present a novel assay that can directly report fusion pores formed between cells ectopically expressing "flipped t-SNAREs" (t-Cell) and nanodiscs (∼17 nm flat bilayers stabilized with the membrane scaffold protein) reconstituted with the cognate v-SNAREs (v-NDs). Currents from a t-Cell-attached membrane patch are recorded. v-NDs that are placed into the patch pipette slowly diffuse to the patched cell surface where outward-facing t-SNAREs are present. Fusion of the v-ND with the cell patch results in a fusion pore connecting the cytoplasm to the pipette solution, whose expansion is prevented by the ND scaffold. This results in a long-lived fusion pore whose size fluctuations are directly related to the measured current fluctuations. These current measurements, reminiscent of single-channel recordings, provide high signal-to-noise ratios and are free of potential artifacts compared with time-resolved admittance and electrochemical measurements. 80-100% of the patched t-Cells had at least one opening (fusion event) with v-NDs, whereas dramatically fewer openings with much smaller amplitudes were observed in control experiments where fusion was inhibited using neurotoxins, protein-free NDs, wild-type cells, or in the presence of the soluble domain of the v-SNARE which competitively binds to available t-SNAREs. Typical openings had currents of a few pA, corresponding to a conductance of ∼400 pS and a pore size of ∼1 nm.