Allosteric modulation of the presynaptic Ca2+ sensor for vesicle fusion

Neurotransmitter release is triggered by an increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i), but it is unknown whether the Ca²⁺-sensitivity of vesicle fusion is modulated during synaptic plasticity. We investigated whether the potentiation of neurotransmitter release by phorbol esters, which target presynaptic protein kinase C (PKC)/munc-13 signalling cascades, exerts a direct effect on the Ca²⁺-sensitivity of vesicle fusion. Using direct presynaptic Ca²⁺-manipulation and Ca²⁺ uncaging at a giant presynaptic terminal, the calyx of Held, we show that phorbol esters potentiate transmitter release by increasing the apparent Ca²⁺-sensitivity of vesicle fusion. Phorbol esters potentiate Ca²⁺-evoked release as well as the spontaneous release rate. We explain both effects by an increased fusion `willingness' in a new allosteric model of Ca²⁺-activation of vesicle fusion. In agreement with an allosteric mechanism, we observe that the classically high Ca²⁺ cooperativity in triggering vesicle fusion (~ 4) is gradually reduced below 3µM [Ca²⁺]_i, reaching a value of <1 at basal [Ca²⁺]_i. Our data indicate that spontaneous transmitter release close to resting [Ca²⁺]_i is a consequence of an intrinsic property of the molecular machinery that mediates synaptic vesicle fusion.