The Mechanism of LTXN4C-Induced Ca2+ Influx Involves Latrophilin-Mediated Activation of Cav2.x Channels

Store-operated Ca²⁺ entry (SOCE) is a key regulator of cytosolic Ca²⁺ (Ca²⁺_cyt). Presynaptic SOCE can be activated by ligands like α-latrotoxin, which acts through the presynaptic G-protein-coupled receptor latrophilin-1 (LPHN1), inducing Ca²⁺ influx and neurotransmitter release. To understand how SOCE-associated proteins contribute to LPHN1 signaling in neurons, we used mouse neuroblastoma NB2a cells as a genetically tractable neuronal model. The cells were stably transfected with exogenous LPHN1 or its non-signaling mutant and stimulated with the non-pore-forming α-latrotoxin mutant LTX^N4C, a known trigger of neurotransmitter release. LPHN1 expression increased the proportion of neuron-like cells and upregulated the voltage-gated Ca²⁺ channels Ca_v1.2 and Ca_v2.1. LPHN1 stimulation by LTX^N4C induced a small Ca²⁺ release sensitive to thapsigargin, and a strong, gradual influx of Ca²⁺, which was insensitive to thapsigargin. Single-cell imaging revealed that this influx consisted of desynchronized high-amplitude Ca²⁺ oscillations in individual cells. This response was reduced by Orai2 knockdown and completely blocked by the Ca_v2.1/2.2 inhibitor ω-conotoxin MVIIC. We conclude that LPHN1 activation by LTX^N4C primes Ca²⁺ stores and induces the opening of Ca_v2.1/2.2 channels. These channels mediate an initial Ca²⁺ influx that triggers Ca²⁺-induced Ca²⁺ release and SOCE. This mechanism, elucidated in model cells, can explain how LTX^N4C stimulates neurotransmitter release.