Abstract
The opening of a fusion pore during exocytosis creates the first aqueous connection between the lumen of a vesicle and the extracellular space. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate the formation of these dynamic structures, and their kinetic transitions are tightly regulated by accessory proteins at the synapse. Here, we utilize two single molecule approaches, nanodisc-based planar bilayer electrophysiology and single-molecule FRET, to address the relationship between SNARE complex assembly and rapid (micro-millisecond) fusion pore transitions, and to define the role of accessory proteins. Synaptotagmin (syt) 1, a major Ca2+-sensor for synaptic vesicle exocytosis, drove the formation of an intermediate: committed trans-SNARE complexes that form large, stable pores. Once open, these pores could only be closed by the action of the ATPase, NSF. Time-resolved measurements revealed that NSF-mediated pore closure occurred via a complex ‘stuttering’ mechanism. This simplified system thus reveals the dynamic formation and dissolution of fusion pores.
Highlights
The opening of a fusion pore during exocytosis creates the first aqueous connection between the lumen of a vesicle and the extracellular space
We reconstituted full-length syt[1], along with syb[2], into NDs (Fig. 1a) and t-sensitive factor attachment protein receptors (SNAREs) into BLMs (Supplementary Fig. 1a); pore formation between the ND and BLM was monitored via the currents that were detected (Fig. 1b)
At present, little is known concerning the structure and dynamics of trans-SNARE complexes because they have been difficult to trap in distinct functional states
Summary
The opening of a fusion pore during exocytosis creates the first aqueous connection between the lumen of a vesicle and the extracellular space. Synaptotagmin (syt) 1, a major Ca2+-sensor for synaptic vesicle exocytosis, drove the formation of an intermediate: committed trans-SNARE complexes that form large, stable pores. Once open, these pores could only be closed by the action of the ATPase, NSF. A key intermediate in this pathway is called the fusion pore, which represents the first aqueous connection between the lumen of a secretory vesicle and the cell exterior[5] These are nanometer-scale transient structures, lasting only milliseconds before they either close or dilate as the vesicle membrane collapses into the plasmalemma[5,6]. This would have ramifications in, for example, kissand-run exocytosis[15]
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