Synaptotagmin-1 and complexin inhibit spontaneous vesicle fusion by masking PIP2, not by clamping SNARE assembly

Vesicle fusion underlies neurotransmitter release, enabling cellular communication. The rapid kinetics of vesicle fusion, tight docking to the plasma membrane, and coordination by multiple SNARE complexes suggest that SNARE complexes are already pre-assembled before fusion. Synaptotagmin-1 (Syt-1) and complexin (CPLX) have been proposed to clamp SNARE assembly and arrest fusion. However, these models are largely based on studies conducted under low ionic strength and structural analyses utilizing SNARE–Syt-1 chimera conjugates. We propose phosphatidylinositol 4,5-bisphosphate (PIP₂) as a critical lipid catalyst that facilitates fusion through electrostatic dehydration. Here we show that neither the C2AB domain of Syt-1 nor CPLX-2 has clamping or inhibitory effect on SNARE assembly. Instead, the C2AB domain and CPLX-2 inhibit Ca²⁺-independent vesicle fusion by masking PIP₂. Our data resolve the long-standing question of increased spontaneous neurotransmitter release in Syt-1 and CPLX knockout neurons, emphasizing PIP₂as an electrostatic lipid catalyst for fusion.