Latrophilin-1-Mediated Gαq Signaling, Store-Operated Ca2+ Entry, and CaV2.1 Activation Control Spontaneous Exocytosis at the Mouse Neuromuscular Junction

Latrophilin 1 (LPHN1/ADGRL1), an adhesion G-protein-coupled receptor (GPCR), is the principal receptor for α-latrotoxin (αLTX), a toxin that triggers massive neurotransmitter release. However, its endogenous signaling mechanism remains elusive. Here, we dissect the LPHN1 signaling pathway at the vertebrate neuromuscular junction, using the pore-deficient αLTX mutant LTXN4C as a selective agonist. Combining electrophysiological recordings from LPHN1 knockout mice with pharmacological inhibitors, calcium imaging, and biochemical assays, we delineate the cascade from receptor activation to spontaneous quantal acetylcholine release. We demonstrate that LPHN1 is specifically localized to the presynaptic membrane and mediates LTXN4C-evoked release. Upon activation, LPHN1 engages the Gαq–phospholipase C pathway to generate inositol 1,4,5-trisphosphate (IP3), triggering Ca2+ release from intracellular stores via IP3 receptors. This store depletion activates store-operated Ca2+ entry (SOCE), providing sustained Ca2+ required for LTXN4C-induced burst-like exocytosis. We uncover distinct roles for CaV2.1 and CaV1 channels in initiating and sustaining this response. These findings establish LPHN1 as a GPCR that harnesses intracellular stores and SOCE to drive spontaneous neurotransmission, revealing a novel signaling paradigm for adhesion GPCRs in presynaptic function.