An ArfGAP-dependent signaling modulates synaptic plasticity via IP3-regulated calcium release from the endoplasmic reticulum

Bhagaban Mallik, Shikha Kushwaha, Anjali Bisht, Harsha Mj, C Andrew Frank and Vimlesh Kumar

PLOS Genetics, 2026, vol. 22, issue 1, 1-33

Abstract: Calcium release from intracellular stores influences synaptic response timing and magnitude. Despite the critical role of inositol trisphosphate (IP3)- and ryanodine receptor (RyR)-dependent calcium release in regulating synaptic strength, the upstream signaling mechanisms that govern IP3 receptor or RyR activity remain elusive. Here, we provide evidence that the ArfGAP-containing protein Asap modulates NMJ morphogenesis and synaptic calcium homeostasis by activating IP3-mediated calcium release from the endoplasmic reticulum (ER) via the phospholipase C-beta (PLCβ) signaling pathway. Using CRISPR/Cas9-engineered Asap mutants and genetically encoded calcium sensors, we demonstrate that loss of Asap leads to elevated resting synaptic calcium, resulting in increased evoked amplitude, elevated spontaneous miniature frequency, and reduced synaptic failures under low extracellular calcium conditions. Additional pharmacological and genetic manipulations of calcium regulatory pathways further support the role of increased resting intracellular calcium in driving enhanced neurotransmission in Asap-deficient synapses. Consistent with the role of Asap’s ArfGAP domain in NMJ morphogenesis and intracellular calcium regulation, expressing a GDP-locked form of Arf6 (Arf6DN) or knocking down Arf6 in Asap mutants not only rescues Asap-associated synaptic defects but also normalizes synaptic calcium levels. Furthermore, epistatic analysis revealed that attenuation of IP3-signaling components in animals constitutively expressing Arf6CA normalized the NMJ morphological defects and synaptic functions. Together, these findings provide novel insights into the role of Asap-Arf6-PLCβ signaling in IP3-regulated calcium dynamics, sustaining both structural and functional synaptic plasticity.Author summary: Calcium signaling plays a central role in how neurons communicate and adapt, yet the upstream mechanisms that control calcium release from internal stores remain poorly understood. In this study, we identify a previously unrecognized signaling pathway that regulates active zone organization and calcium dynamics at the Drosophila neuromuscular junctions. We show that the ArfGAP-domain containing protein Asap acts through the small GTPase Arf6 and phospholipase C-beta (PLCβ) to stimulate inositol trisphosphate (IP3)-dependent calcium release from the endoplasmic reticulum. Loss of Asap disrupts calcium homeostasis, leading to elevated basal calcium levels, increased neurotransmitter release, and structural changes at synaptic terminals. Suppressing Arf6 activity or blocking IP3 signaling restores normal synaptic structure and function, highlighting a critical role for the Asap-Arf6-PLCβ-IP3 pathway in maintaining basal synaptic calcium balance, active zone integrity and synaptic stability. These findings reveal a new molecular link between small GTPases and calcium signaling mechanisms that fine-tune synaptic communication.

Date: 2026
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Persistent link: https://EconPapers.repec.org/RePEc:plo:pgen00:1012031

DOI: 10.1371/journal.pgen.1012031

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