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

Previous studies suggest that the vesicular cysteine-string protein (CSP) m ay modulate presynaptic Ca2+ channel activity in fast neurotransmitter rele ase. To test this hypothesis, we analyzed the dynamics of presynaptic Ca2ion influx with the Ca2+ indicator fluo-4 AM at csp mutant neuromuscular ju nctions of Drosophila. From 24 to 30 degrees C, stimulus-evoked, relative p resynaptic Ca2+ signals were increasingly larger in csp mutant boutons than in controls. Above 30 degrees C, Ca2+ signals declined and were similar to controls at 34 degrees C. A prolonged decay of Ca2+ signals in mutant bout ons at high temperatures indicated abnormally slow Ca2+ clearance. Cytosoli c Ca2+ at rest was determined with the ratiometric Ca2+ indicator fura-2 AM and was similar in mutant and control boutons at 24 degrees C but higher i n mutant boutons at 34 degrees C. Despite larger Ca2+ signals in mutant bou tons, evoked neurotransmitter release was always reduced in csp mutants and exhibited pronounced facilitation. Thus, a lack of Ca2+ entry cannot expla in the reduction of neurotransmitter release in csp mutants. At all tempera tures tested, raising extracellular Ca2+ increased transmitter release elic ited by single stimuli in csp mutants. Collectively, these data suggest mul tiple functions for CSP at synaptic terminals. Increased Ca2+ signals coupl ed with reduced release suggest a direct function of CSP in exocytosis down stream from Ca2+ entry. Because the reduction of evoked release in csp muta nts is counteracted by increased Ca2+ levels, we suggest that CSP primarily increases the Ca2+ sensitivity of the exocytotic machinery.