Norepinephrine exocytosis stimulated by alpha-latrotoxin requires both external and stored Ca2+ and is mediated by latrophilin, G proteins and phospholipase C

alpha-latrotoxin (LTX) stimulates massive release of neurotransmitters by b inding to a heptahelical transmembrane protein, latrophilin. Our experiment s demonstrate that latrophilin is a G-protein-coupled receptor that specifi cally associates with heterotrimeric G proteins. The latrophilin-G protein complex is very stable in the presence of GDP but dissociates when incubate d with GTP, suggesting a functional interaction. As revealed by immunostain ing, latrophilin interacts with G alpha(q/11) and G alpha(0) but not with G alpha(s), G alpha(i) or G alpha(z), indicating that this receptor may coup le to several G proteins but it is not promiscuous. The mechanisms underlyi ng LTX-evoked norepinephrine secretion from rat brain nerve terminals were also studied. In the presence of extracellular Ca2+, LTX triggers vesicular exocytosis because botulinum neurotoxins E, Cl or tetanus toxin inhibit th e Ca2+-dependent component of the toxin-evoked release. Based on (i) the kn own involvement of G alpha(q) in the regulation of inositol-1,4,5-triphosph ate generation and (ii) the requirement for Ca2+ in LTX action, we tested t he effect of inhibitors of Ca2+ mobilization on the toxin-evoked norepineph rine release. It was found that aminosteroid U73122, which inhibits the cou pling of G proteins to phospholipase C, blocks the Ca2+ dependent toxin's a ction. Thapsigargin, which depletes intracellular Ca2+ stores, also potentl y decreases the effect of LTX in the presence of extracellular Ca2+ On the other hand, clostridial neurotoxins or drugs interfering with Ca2+ metaboli sm do not inhibit the Ca2+-independent component of LTX-stimulatcd release. In the absence of Ca2+, the toxin induces in the presynaptic membrane non- selective pores permeable to small fluorescent dyes; these pores may allow efflux of neurotransmitters from the cytoplasm. Our results suggest that LT X stimulates norepinephrine exocytosis only in the presence of external Ca2 + provided intracellular Ca2+ stores are unperturbed and that latrophilin, G proteins and phospholipase C may mediate the mobilization of stored Ca2+, which then triggers secretion.