Dilation of Fusion Pores by SNARE Protein Crowding

Abstract

Hormones and neurotransmitters are released through exocytotic fusion pores that can fluctuate in size and flicker open and shut multiple times. The kinetics and the amount of cargo released, and the mode of vesicle recycling depend on the fate of the pore, which may reseal or dilate irreversibly. Pore nucleation requires zippering between vesicle-associated v- and target membrane t-SNAREs, but the mechanisms governing the subsequent pore dilation are not known. Past approaches either monitored single exocytotic pores in live cells with unknown biochemistry, or used reconstitutions that lacked single pore sensitivity. Here, we probed dilation of single fusion pores using v-SNARE-reconstituted ∼23 nm diameter discoidal nanolipoprotein particles (vNLPs) as fusion partners with cells ectopically expressing cognate, “flipped” t-SNAREs. A flipped t-SNARE cell is patch-clamped in the cell-attached configuration with the vNLPs included in the pipette solution. Fusion of a vNLP with the cell surface produces a pore connecting the cytosol with the pipette solution, through which direct-currents are measured under voltage clamp. The magnitude of the current reports pore size with sub-millisecond time resolution (Wu, Z. et al. Sci. Rep. 2016). We found that pore nucleation required a minimum of 2, and reached a maximum above ∼4 copies of v-SNAREs per NLP face. In contrast, the mean conductance of single pores increased as copy number was increased and was far from saturating at 15 copies, the NLP capacity. Thus, very different numbers of SNARE complexes cooperate at the distinct stages of fusion pore nucleation and pore dilation. We calculated pore size distributions and free energy profiles versus pore size. Combined with a mathematical model, these results suggest crowding of SNARE complexes at the pore waist drive pore expansion.