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

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.

Hypothesis – buttressed rings assemble, clamp, and release SNAREpins for synaptic transmission

“…As they get closer, they exert their force over an ever‐smaller area, and pressure rises geometrically to reach 400–500 atm when their zippering tips circumscribe a disc of ~ 4 nm diameter (Appendix 1). This will create an initial fusion pore opening no larger than ~ 2 nm in diameter consistent with experimental observations 55, 58. This should require < 1 ms (Appendix 2).…”

Dilation of fusion pores by crowding of SNARE proteins

“…We confirmed that vNLPs fused with liposomes that were reconstituted with t-SNAREs in a SNARE-dependent manner using a previously described bulk assay that monitors calcium release through pores connecting v-SNARE nanodiscs with t-SNARE liposomes (Bello et al, 2016; Shi et al, 2012) (Figure 1—figure supplement 1a). Although NLP pores could in principle grow to $\gtrsim 10$ nm in diameter (Figure 4b), much larger than the $~4$ nm allowed by the membrane scaffold protein (MSP) based small ND geometry (Wu et al, 2016; Shi et al, 2012), bulk calcium release rates were comparable between vNLP and vMSP NDs loaded with similar v-SNARE copy numbers (Figure 1—figure supplement 1b), confirming that the bulk assay is largely insensitive to pore properties under these conditions (Bello et al, 2016).

10.7554/eLife.22964.002Figure 1.Size separation and characterization of NLPs.( a ) Representative size exclusion chromatograms for various NLP preparations as indicated.

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“…NLPs that were reconstituted with eight copies of the complementary neuronal v-SNARE VAMP2/synaptobrevin (vNLPs) were included in the pipette solution (100 nM vNLPs, 120 µM lipid); they diffused to the pipette tip and fused with the patch. Because an NLP is not a closed structure like a vesicle, its fusion with the voltage-clamped membrane patch establishes a direct conduction pathway between the cytosol and the pipette solution, leading to direct-currents whose magnitude reflects pore geometry (Wu et al, 2016).

10.7554/eLife.22964.008Figure 4.Detection of single-pores between vNLP nanodiscs and tCells.( a, b ) Schematic of the assay.

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The Multifaceted Role of SNARE Proteins in Membrane Fusion

“…The zippering of the TMDs of t- and v-SNARE proteins facilitated lipid mixing and pore nucleation and the presence of the heterodimer at the pore rim was suggested to obstruct pore resealing (Wu et al, 2016). …”