Cysteine-String Protein Increases the Calcium Sensitivity of Neurotransmitter Exocytosis in Drosophila

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Abstract
Previous studies suggest that the vesicular cysteine-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 cspmutants 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 cspmutants. 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.