title: Norepinephrine exocytosis stimulated by alpha-latrotoxin requires both external and stored Ca2+ and is mediated by latrophilin, G proteins and phospholipase C.
abstract: alpha-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 G alpha q/11 and G alpha o but not with G alpha s, G alpha i or G alpha 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 Ca2+, LTX triggers vesicular exocytosis because botulinum neurotoxins E, Cl or tetanus toxin inhibit the Ca(2+)-dependent component of the toxin-evoked release. Based on (i) the known involvement of G alpha q in the regulation of inositol-1,4,5-triphosphate generation and (ii) the requirement for 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 Ca(2+)-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.
authors.affiliation: Biochemistry Department, Imperial College, London, UK.
authors.forename: M A
authors.initials: MA
authors.lastname: Rahman
authors.forename: A C
authors.initials: AC
authors.lastname: Ashton
authors.forename: F A
authors.initials: FA
authors.lastname: Meunier
authors.forename: B A
authors.initials: BA
authors.lastname: Davletov
authors.forename: J O
authors.initials: JO
authors.lastname: Dolly
authors.forename: Y A
authors.initials: YA
authors.lastname: Ushkaryov
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology / Q000502:physiology; D005089:Exocytosis / Q000187:drug effects* / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000502:physiology; D066298:In Vitro Techniques; D009638:Norepinephrine / Q000378:metabolism*; D051381:Rats; D018000:Receptors, Peptide / Q000502:physiology; D013111:Spider Venoms / Q000494:pharmacology*; D013574:Synaptosomes / Q000187:drug effects / Q000502:physiology; D010738:Type C Phospholipases / Q000502:physiology
chemical_list: D018000:Receptors, Peptide; D013111:Spider Venoms; C034800:alpha-latrotoxin receptor; C020763:alpha-latrotoxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium; D009638:Norepinephrine