Phosphatidylinositol phosphates as co-activators of Ca²⁺ binding to C₂ domains of synaptotagmin 1

Ca²⁺-dependent phospholipid binding to the C₂A and C₂B domains of synaptotagmin 1 is thought to trigger fast neurotransmitter release, but only Ca₂₊ binding to the C₂B domain is essential for release. To investigate the underlying mechanism, we have compared the role of basic residues in Ca²⁺/phospholipid binding and in release. Mutations in a polybasic sequenceonthe side of the C ₂B domain β-sandwich or in a basic residue in a top Ca ²⁺-binding loop of the C₂A domain (R233) cause comparable decreases in the apparent Ca²⁺ affinity of synaptotagmin 1 and the Ca²⁺ sensitivity of release, whereas mutation of the residue homologous to Arg²³³ in the C₂B domain (Lys³⁶⁶) has no effect. Phosphatidylinositol polyphosphates co-activate Ca ²⁺-dependent and -independent phospholipid binding to synaptotagmin 1, but the effects of these mutations on release only correlate with their effects on the Ca²⁺-dependent component. These results reveal clear distinctions in the Ca²⁺-dependent phospholipid binding modes of the synaptotagmin 1 C₂ domains that may underlie their functional asymmetry and suggest that phosphatidylinositol polyphosphates may serve as physiological modulators of Ca²⁺ affinity of synaptotagmin 1 in vivo.