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 LTXN4C, a known trigger of neurotransmitter release. LPHN1 expression increased the proportion of neuron-like cells and upregulated the voltage-gated Ca2+ channels Cav1.2 and Cav2.1. LPHN1 stimulation by LTXN4C 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 Cav2.1/2.2 inhibitor ω-conotoxin MVIIC. We conclude that LPHN1 activation by LTXN4C primes Ca2+ stores and induces the opening of Cav2.1/2.2 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 LTXN4C stimulates neurotransmitter release.