Phosphatidylinositol 4,5-Bisphosphate Increases Ca2+ Affinity of Synaptotagmin-1 by 40-fold

Synaptotagmin-1 is the main Ca2+ sensor of neuronal exocytosis. It binds to both Ca2+ and the anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), but the precise cooperativity of this binding is still poorly understood. Here, we used microscale thermophoresis to quantify the cooperative binding of PIP2 and Ca2+ to synaptotagmin-1. We found that PIP2 bound to the well conserved polybasic patch of the C2B domain with an apparent dissociation constant of similar to 20 mu M. PIP2 binding reduced the apparent dissociation constant for Ca2+ from similar to 250 to <5 mu M. Thus, our data show that PIP2 makes synaptotagmin-1 >40-fold more sensitive to Ca2+. This interplay between Ca2+, synaptotagmin-1, and PIP2 is crucial for neurotransmitter release.