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