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

M. Atiqur Rahman , Anthony C. Ashton , Frédéric A. Meunier , Bazbek A. Davletov , J. Oliver Dolly , Yuri A. Ushkaryov

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 Gαq/11 and Gαo but not with Gαs, Gαi or Gαz, 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 Ca2+, LTX triggers vesicular exocytosis because botulinum neurotoxins E, C1 or tetanus toxin inhibit the Ca2+–dependent component of the toxin–evoked release. Based on (i) the known involvement of Gαq in the regulation of inositol–1,4,5–triphosphate generation and (ii) the requirement of Ca2+ in LTX action, we tested the effect of inhibitors of Ca2+ 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 Ca2+–dependent toxin's action. Thapsigargin, which depletes intracellular Ca2+ stores, also potently decreases the effect of LTX in the presence of extracellular Ca2+. On the other hand, clostridial neurotoxins or drugs interfering with Ca2+ metabolism do not inhibit the Ca2+–independent component of LTX–stimulated 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 LTX 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.

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