An Alien Divalent Ion Reveals a Major Role for Ca2+ Buffering in Controlling Slow Transmitter Release

Ca2+-dependent transmitter release occurs in a fast and in a slow phase, but the differential roles of Ca2+ buffers and Ca2+ sensors in shaping release kinetics are still controversial. Replacing extracellular Ca2+ by Sr2+ causes decreased fast release but enhanced slow release at many synapses. Here, we established presynaptic Sr2+ uncaging and made quantitative Sr2+ - and Ca2+ -imaging experiments at the mouse calyx of Held synapse, to reveal the interplay between Ca2+ sensors and Ca2+ buffers in the control of fast and slow release. We show that Sr2+ activates the fast, Synaptotagmin-2 (Syt2) sensor for vesicle fusion with sixfold lower affinity but unchanged high cooperativity. Surprisingly, Sr2+ also activates the slow sensor that remains in Syt2 knock-out synapses with a lower efficiency, and Sr2+ was less efficient than Ca2+ in the limit of low concentrations in wild-type synapses. Quantitative imaging experiments show that the buffering capacity of the nerve terminal is markedly lower for Sr2+ than for Ca2+ (similar to 5-fold). This, together with an enhanced Sr2+ permeation through presynaptic Ca2+ channels (similar to 2-fold), admits a drastically higher spatially averaged Sr2+ transient compared with Ca2+. Together, despite the lower affinity of Sr2+ at the fast and slow sensors, the massively higher amplitudes of spatially averaged Sr2+ transients explain the enhanced late release. This also allows us to conclude that Ca2+ buffering normally controls late release and prevents the activation of the fast release sensor by residual Ca2+.