A gain-of-function mutation in synaptotagmin-1 reveals a critical role of Ca²⁺-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex binding in synaptic exocytosis

Synaptotagmin-1, the Ca²⁺ sensor for fast neurotransmitter release, was proposed to function by Ca²⁺-dependent phospholipid binding and/or by Ca²⁺-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex binding. Extensive in vivo data support the first hypothesis, but testing the second hypothesis has been difficult because no synaptotagmin-1 mutation is known that selectively interferes with SNARE complex binding. Using knock-in mice that carry aspartate-to-asparagine substitutions in a Ca²⁺-binding site of synaptotagmin-1 (the D232N or D238N substitutions), we now show that the D232N mutation dramatically increases Ca²⁺-dependent SNARE complex binding by native synaptotagmin-1, but leaves phospholipid binding unchanged. In contrast, the adjacent D238N mutation does not significantly affect SNARE complex binding, but decreases phospholipid binding. Electrophysiological recordings revealed that the D232N mutation increased Ca²⁺-triggered release, whereas the D238N mutation decreased release. These data establish that fast vesicle exocytosis is driven by a dual Ca²⁺-dependent activity of synaptotagmin-1, namely Ca²⁺-dependent binding both to SNARE complexes and to phospholipids.