Synaphin/complexin is a cytosolic protein that preferentially binds to syntaxin within the SNARE complex and promotes SNARE complex oligomerization into higher order structures.

Injection of an inhibitory peptide from the syntaxin-binding domain of complexin into squid giant presynaptic terminals inhibited neurotransmitter release at a late prefusion step of synaptic vesicle exocytosis.

Oligomerization of SNARE complexes into a higher order structure creates a SNARE scaffold for efficient, regulated fusion of synaptic vesicles.

Crystal structure at 2.95 angstrom resolution shows a helical segment of squid complexin (residues 25-98) binds in antiparallel fashion to the SNARE four-helix bundle, contacting syntaxin and synaptobrevin at the ionic zero layer.

The structure is part of a multiprotein fusion machinery that regulates vesicle fusion at a late pre-fusion stage. Ca2+ may initiate membrane fusion by acting directly or indirectly on complexin.

Complexin's binding position on the SNARE bundle strategically prevents the final zippering of the SNAREs at the membrane-proximal end, blocking fusion until Ca2+ signaling occurs.

The accessory-central helical domains of complexin are essential for its inhibitory (clamping) function, and also contribute to rapid Ca2+-synchronized vesicle release.

Complexin and synaptotagmin-1 synergistically clamp SNARE assembly, maintaining a pool of docked vesicles ready for fast release upon Ca2+ influx.

The C-terminal membrane-binding domain of complexin aids clamping by tethering complexin to membranes, increasing local concentration at release sites.

Complexin knockdown in mouse neurons causes a 3-4 fold increase in miniature neurotransmitter release frequency (enhanced spontaneous vesicle fusion).

Complexin knockdown reduces evoked EPSC amplitude by ~3-4 fold and specifically abolishes fast synchronous release triggered by action potentials.

Deletion of the N-terminal 26 amino acids abolishes complexin's ability to synchronize rapid release without affecting its SNARE-binding or baseline clamp.

Four disease-associated CPLX1 mutations have been reported: two nonsense (E108*, C105*), one frameshift (D23Rfs*69), and one missense (L128M), causing infantile epileptic encephalopathies classified as SNAREopathies.

Priming of a vesicle is completed when SNAREs assemble and complexin binds to the SNARE complex, preventing it from disassembling or fully fusing the membranes.

Ca2+ binding to synaptotagmin-1 releases the clamp and allows SNARE complexes to rapidly complete zippering and drive membrane fusion.