Nanodisc-cell fusion: control of fusion pore nucleation and lifetimes by SNARE protein transmembrane domains

Sci Rep. 2016 Jun 6:6:27287. doi: 10.1038/srep27287.

Abstract

The initial, nanometer-sized 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 release and vesicle recycling kinetics, but pore properties are poorly known because biochemically defined single-pore assays are lacking. We isolated single flickering pores connecting v-SNARE-reconstituted nanodiscs to cells ectopically expressing cognate, "flipped" t-SNAREs. Conductance through single, voltage-clamped fusion pores directly reported sub-millisecond pore dynamics. Pore currents fluctuated, transiently returned to baseline multiple times, and disappeared ~6 s after initial opening, as if the fusion pore fluctuated in size, flickered, and resealed. We found that interactions between v- and t-SNARE transmembrane domains (TMDs) promote, but are not essential for pore nucleation. Surprisingly, TMD modifications designed to disrupt v- and t-SNARE TMD zippering prolonged pore lifetimes dramatically. We propose that the post-fusion geometry of the proteins contribute to pore stability.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calcium / metabolism
  • Cell Fusion / methods*
  • Cell Nucleus / metabolism*
  • Exocytosis
  • HeLa Cells
  • Humans
  • Membrane Fusion
  • Neurotransmitter Agents
  • Protein Binding
  • Protein Domains
  • SNARE Proteins / chemistry*
  • SNARE Proteins / metabolism*
  • Secretory Vesicles / metabolism

Substances

  • Neurotransmitter Agents
  • SNARE Proteins
  • Calcium