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Keywords:

  • Ca2+channels;
  • Drosophila;
  • endocytosis;
  • synaptic transmission

Abstract

Endocytosis at the presynaptic terminal is initiated by Ca2+ influx through voltage-gated Ca2+ channels. At the Drosophila neuromuscular junction, we demonstrated two components of endocytosis linked to distinct Ca2+ channels. A voltage-gated Ca2+ channel blocker, (R)-(+)-Bay K8644 (R-BayK), selectively blocked one component (R-BayK-sensitive component) without affecting exocytosis, while low concentrations of La3+ preferentially depressed the other component (La3+ -sensitive component). In a temperature-sensitive mutant, shibirets, at non-permissive temperatures, dynamin clusters were found immunohistochemically at the active zone (AZ) during the R-BayK-sensitive endocytosis, while they were detected at the non-AZ during the La3+-sensitive endocytosis. Immunostaining of the Ca2+ channel α2δ subunit encoded by straightjacket (stj) was found within the AZ, and a mutation in stj depressed the R-BayK-sensitive component but enhanced the La3+ -sensitive one, indicating that the α2δ subunit is associated with the R-BayK-sensitive Ca2+ channel. Filipin bound to the non-AZ membrane and inhibited the La3+ -sensitive component, but not the R-BayK-sensitive one. We concluded that the R-BayK-sensitive component of endocytosis occurred at the AZ and termed this AZ endocytosis. We also concluded that the La3+ -sensitive component occurred at the non-AZ and termed this non-AZ endocytosis. These two types of endocytosis were modulated by various drugs towards opposite directions, indicating that they were differentially regulated. During high-frequency stimulation, AZ endocytosis operated mainly in the early phase, whereas non-AZ endocytosis operated in the late phase. Thus, intense synaptic transmission is coordinately maintained by synaptic vesicle recycling initiated by Ca2+ influx through the two types of Ca2+ channel.