Cysteine-string protein increases the calcium sensitivity of neurotransmitter exocytosis in Drosophila

Previous studies suggest that the vesicular s-string protein (CSP) may modulate presynaptic Ca²⁺ channel activity in fast neurotransmitter release. To test this hypothesis, we analyzed the dynamics of presynaptic Ca²⁺ ion influx with the Ca²⁺ indicator fluo-4 AM at csp mutant neuromuscular junctions of Drosophila. From 24 to 30°C, stimulus-evoked, relative presynaptic Ca²⁺ signals were increasingly larger in csp mutant boutons than in controls. Above 30°C, Ca²⁺ signals declined and were similar to controls at 34°C. A prolonged decay of Ca²⁺ signals in mutant boutons at high temperatures indicated abnormally slow Ca²⁺ clearance. Cytosolic Ca²⁺ at rest was determined with the ratiometric Ca²⁺ indicator fura-2 AM and was similar in mutant and control boutons at 24°C but higher in mutant boutons at 34°C. Despite larger Ca²⁺ signals in mutant boutons, evoked neurotransmitter release was always reduced in csp mutants and exhibited pronounced facilitation. Thus, a lack of Ca²⁺ entry cannot explain the reduction of neurotransmitter release in csp mutants. At all temperatures tested, raising extracellular Ca²⁺ increased transmitter release elicited by single stimuli in csp mutants. Collectively, these data suggest multiple functions for CSP at synaptic terminals. Increased Ca²⁺ signals coupled with reduced release suggest a direct function of CSP in exocytosis downstream from Ca²⁺ entry. Because the reduction of evoked release in csp mutants is counteracted by increased Ca²⁺ levels, we suggest that CSP primarily increases the Ca²⁺ sensitivity of the exocytotic machinery.