Cysteine-string protein increases the calcium sensitivity of neurotransmitter exocytosis in Drosophila.
Previous studies suggest that the vesicular cysteine-string protein (CSP) may modulate presynaptic Ca(2+) channel activity in fast neurotransmitter release. To test this hypothesis, we analyzed the dynamics of presynaptic Ca(2+) ion influx with the Ca(2+) indicator fluo-4 AM at csp mutant neuromuscular junctions of Drosophila. From 24 to 30 degrees C, stimulus-evoked, relative presynaptic Ca(2+) signals were increasingly larger in csp mutant boutons than in controls. Above 30 degrees C, Ca(2+) signals declined and were similar to controls at 34 degrees C. A prolonged decay of Ca(2+) signals in mutant boutons at high temperatures indicated abnormally slow Ca(2+) clearance. Cytosolic Ca(2+) at rest was determined with the ratiometric Ca(2+) indicator fura-2 AM and was similar in mutant and control boutons at 24 degrees C but higher in mutant boutons at 34 degrees C. Despite larger Ca(2+) signals in mutant boutons, evoked neurotransmitter release was always reduced in csp mutants and exhibited pronounced facilitation. Thus, a lack of Ca(2+) entry cannot explain the reduction of neurotransmitter release in csp mutants. At all temperatures tested, raising extracellular Ca(2+) increased transmitter release elicited by single stimuli in csp mutants. Collectively, these data suggest multiple functions for CSP at synaptic terminals. Increased Ca(2+) signals coupled with reduced release suggest a direct function of CSP in exocytosis downstream from Ca(2+) entry. Because the reduction of evoked release in csp mutants is counteracted by increased Ca(2+) levels, we suggest that CSP primarily increases the Ca(2+) sensitivity of the exocytotic machinery.
Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6974, USA.
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Dawson-Scully
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Bronk
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Atwood
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Zinsmaier
D000818:Animals; D001831:Body Temperature / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism*; D004558:Electric Stimulation; D005089:Exocytosis / Q000502:physiology*; D050956:HSP40 Heat-Shock Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000502:physiology; D009469:Neuromuscular Junction / Q000378:metabolism / Q000648:ultrastructure; D018377:Neurotransmitter Agents / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000378:metabolism / Q000648:ultrastructure
D015220:Calcium Channels; D050956:HSP40 Heat-Shock Proteins; D008565:Membrane Proteins; D018377:Neurotransmitter Agents; C088460:cysteine string protein; D002118:Calcium