Norepinephrine exocytosis stimulated by α–latrotoxin requires both external and stored Ca 2+ and is mediated by latrophilin, G proteins and phospholipase C

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Abstract
α–latrotoxin (LTX) stimulates massive release of neurotransmitters by binding to a heptahelical transmembrane protein, latrophilin. Our experiments demonstrate that latrophilin is a G–protein–coupled receptor that specifically associates with heterotrimeric G proteins. The latrophilin–G protein complex is very stable in the presence of GDP but dissociates when incubated with GTP, suggesting a functional interaction. As revealed by immunostaining, latrophilin interacts with Gα q/11 and Gα o but not with Gα s , Gα i or Gα z , indicating that this receptor may couple to several G proteins but it is not promiscuous. The mechanisms underlying LTX–evoked norepinephrine secretion from rat brain nerve terminals were also studied. In the presence of extracellular Ca 2+ , LTX triggers vesicular exocytosis because botulinum neurotoxins E, C1 or tetanus toxin inhibit the Ca 2+ –dependent component of the toxin–evoked release. Based on (i) the known involvement of Gα q in the regulation of inositol–1,4,5–triphosphate generation and (ii) the requirement of Ca 2+ in LTX action, we tested the effect of inhibitors of Ca 2+ mobilization on the toxin–evoked norepinephrine release. It was found that aminosteroid U73122, which inhibits the coupling of G proteins to phospholipase C, blocks the Ca 2+ –dependent toxin's action. Thapsigargin, which depletes intracellular Ca 2+ stores, also potently decreases the effect of LTX in the presence of extracellular Ca 2+ . On the other hand, clostridial neurotoxins or drugs interfering with Ca 2+ metabolism do not inhibit the Ca 2+ –independent component of LTX–stimulated release. In the absence of Ca 2+ , 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 LTX stimulates norepinephrine exocytosis only in the presence of external Ca 2+ provided intracellular Ca 2+ stores are unperturbed and that latrophilin, G proteins and phospholipase C may mediate the mobilization of stored Ca 2+ , which then triggers secretion.