Presynaptic PMCA interacts with Dlg, regulating in vivo synaptic calcium in Drosophila larval NMJ

Precise regulation of presynaptic Ca2+is essential for neurotransmitter release and synaptic function. At the Drosophila larval neuromuscular junction (NMJ), the plasma membrane Ca2+-ATPase (PMCA) mediates presynaptic Ca2+; extrusion and is enriched in peri-active-zone membrane domains. Here, we investigated how PMCA distribution is regulated and contributes to synaptic function. Co-immunoprecipitation and super-resolution STED microscopy revealed that PMCA interacts and co-localizes with the MAGUK scaffold protein Discs large (Dlg) in discrete presynaptic membrane clusters. Neuronal depletion of the DlgS97 isoform disrupted PMCA clustering without substantially altering its abundance, redistributing the pump into smaller membrane puncta. Reporter assays further showed that PDZ-binding motifs in the PMCA C-terminus mediate its interaction with Dlg and are required for normal cluster formation. Electrophysiological analyses revealed that reducing either PMCA or Dlg increased spontaneous neurotransmitter release and enhanced asynchronous release following high-frequency stimulation. PMCA depletion also prolonged postsynaptic current decay during repetitive stimulation, implicating PMCA in the rapid regulation of presynaptic Ca2+; signals. Together, these findings identify Dlg as a key organizer of presynaptic PMCA and establish a link between synaptic scaffold organization and Ca2+; homeostasis in vivo. Our results demonstrate that PMCA contributes to maintaining resting presynaptic Ca2+; levels, restoring Ca2+; homeostasis after repetitive stimulation, and regulating Ca2+; dynamics following a single stimulus. These findings highlight the importance of the nanoscale organization of Ca2+; extrusion machinery in controlling neurotransmitter release at the Drosophila NMJ.