Phospholipids Differentially Regulate Ca²⁺ Binding to Synaptotagmin-1

Synaptotagmin-1 (Syt-1) is a calcium sensing protein that is resident in synaptic vesicles. It is well established that Syt-1 is essential for fast and synchronous neurotransmitter release. However, the role of Ca²⁺ and phospholipid binding in the function of Syt-1, and ultimately in neurotransmitter release, is unclear. Here, we investigate the binding of Ca²⁺ to Syt-1, first in the absence of lipids, using native mass spectrometry to evaluate individual binding affinities. Syt-1 binds to one Ca²⁺ with a K_D ∼ 45 μM. Each subsequent binding affinity (n ≥ 2) is successively unfavorable. Given that Syt-1 has been reported to bind anionic phospholipids to modulate the Ca²⁺ binding affinity, we explored the extent that Ca²⁺ binding was mediated by selected anionic phospholipid binding. We found that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and dioleoylphosphatidylserine (DOPS) positively modulated Ca²⁺ binding. However, the extent of Syt-1 binding to phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂) was reduced with increasing [Ca²⁺]. Overall, we find that specific lipids differentially modulate Ca²⁺ binding. Given that these lipids are enriched in different subcellular compartments and therefore may interact with Syt-1 at different stages of the synaptic vesicle cycle, we propose a regulatory mechanism involving Syt-1, Ca²⁺, and anionic phospholipids that may also control some aspects of vesicular exocytosis.