Calcium accelerates endocytosis of vSNAREs at hippocampal synapses.

A pH-sensitive form of green-fluorescent protein (GFP) fused to the lumenal domain of VAMP (synapto-pHluorin) provides a sensitive optical probe to track the net balance between exocytosis and endocytosis of this protein at small synaptic terminals of the central nervous system. Here we used a reversible proton-pump blocker that prevents vesicle re-acidification upon endocytosis to trap vesicles in the alkaline state during recycling. In combination with optical measurements of synapto-pHluorin, we used alkaline trapping to examine the kinetic components of exocytosis and endocytosis separately at synaptic terminals. Using this approach, we show that, in addition to controlling exocytosis, intracellular calcium levels tightly regulate the speed of endocytosis, increasing it to a maximal speed of approximately one vesicle per second.