Structural studies of complexin bound to a mimetic of a prefusion SNARE complex provide insight into how complexin 'clamps' SNARE complex assembly, thereby inhibiting membrane fusion. While the central helix of complexin is anchored to a SNARE complex, its accessory helix extends away and bridges to a second SNARE complex, generating a zigzag array incompatible with fusion. Complexin prevents SNAREs from releasing neurotransmitters until an action potential arrives at the synapse. To understand the mechanism for this inhibition, we determined the structure of complexin bound to a mimetic of a prefusion SNAREpin lacking the portion of the v-SNARE that zippers last to trigger fusion. The 'central helix' of complexin is anchored to one SNARE complex, while its 'accessory helix' extends away at ~45° and bridges to a second complex, occupying the vacant v-SNARE binding site to inhibit fusion. We expected the accessory helix to compete with the v-SNARE for t-SNARE binding but found instead that the interaction occurs intermolecularly. Thus, complexin organizes the SNAREs into a zigzag topology that, when interposed between the vesicle and plasma membranes, is incompatible with fusion.

Hearing relies on rapid, temporally precise, and sustained neurotransmitter release at the ribbon synapses of sensory cells, the inner hair cells (IHCs). This process requires otoferlin, a six C₂-domain, Ca²⁺-binding transmembrane protein of synaptic vesicles. To decipher the role of otoferlin in the synaptic vesicle cycle, we produced knock-in mice (Otof ^{Ala515,Ala517/Ala515,Ala517}) with lower Ca²⁺-binding affinity of the C₂C domain. The IHC ribbon synapse structure, synaptic Ca²⁺ currents, and otoferlin distribution were unaffected in these mutant mice, but auditory brainstem response wave-I amplitude was reduced. Lower Ca²⁺ sensitivity and delay of the fast and sustained components of synaptic exocytosis were revealed by membrane capacitance measurement upon modulations of intracellular Ca²⁺ concentration, by varying Ca²⁺ influx through voltage-gated Ca²⁺-channels or Ca²⁺ uncaging. Otoferlin thus functions as a Ca²⁺ sensor, setting the rates of primed vesicle fusion with the presynaptic plasma membrane and synaptic vesicle pool replenishment in the IHC active zone.

Significance: Synaptotagmin-1 is the calcium sensor for synchronous neurotransmitter release. It couples calcium influx to the soluble N -ethylmaleimide–sensitive factor activating protein receptor (SNARE)-catalyzed fusion, but how this coupling happens is unknown. Here, using electron microscopy, we report that the cytosolic domain of synaptotagmin can assemble into ring-like oligomers under calcium-free conditions, and these rings disassemble rapidly upon calcium binding. This process suggests a novel but speculative mechanism to explain calcium coupling, in which the synaptotagmin rings separate the vesicle and plasma membranes and prevent the completion of SNARE complex assembly until the influx of calcium.

Hormones and neurotransmitters are released through fluctuating exocytotic fusion pores that can flicker open and shut multiple times. Cargo release and vesicle recycling depend on the fate of the pore, which may reseal or dilate irreversibly. Pore nucleation requires zippering between vesicle-associated v-SNAREs and target membrane t-SNAREs, but the mechanisms governing the subsequent pore dilation are not understood. Here, we probed the 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. Pore nucleation required a minimum of two v-SNAREs per NLP face, and further increases in v-SNARE copy numbers did not affect nucleation rate. By contrast, the probability of pore dilation increased with increasing v-SNARE copies and was far from saturating at 15 v-SNARE copies per face, the NLP capacity. Our experimental and computational results suggest that SNARE availability may be pivotal in determining whether neurotransmitters or hormones are released through a transient ('kiss and run') or an irreversibly dilating pore (full fusion).