Synaptotagmin-1 functions as a Ca²⁺ sensor for spontaneous release

Spontaneous 'mini' release occurs at all synapses, but its nature remains enigmatic. We found that 95% of spontaneous release in murine cortical neurons was induced by Ca 2⁺-binding to synaptotagmin-1 (Syt1), the Ca 2 ⁺ sensor for fast synchronous neurotransmitter release. Thus, spontaneous and evoked release used the same Ca 2+ -dependent release mechanism. As a consequence, Syt1 mutations that altered its Ca 2+ affinity altered spontaneous and evoked release correspondingly. Paradoxically, Syt1 deletions (as opposed to point mutations) massively increased spontaneous release. This increased spontaneous release remained Ca 2⁺ dependent but was activated at lower Ca 2⁺concentrations and with a lower Ca 2 ⁺ cooperativity than synaptotagmin-driven spontaneous release. Thus, in addition to serving as a Ca 2⁺ senso r for spontaneous and evoked release, Syt1 clamped a second, more sensitive Ca 2⁺ sensor for spontaneous release that resembles the Ca 2⁺ sensor for evoked asynchronous release. These data suggest that Syt1 controls both evoked and spontaneous release at a synapse as a simultaneous Ca 2⁺ -dependent activator and clamp of exocytosis.