Skip to main content
NCBI home page
Philosophical Transactions of the Royal Society B: Biological Sciences logoLink to Philosophical Transactions of the Royal Society B: Biological Sciences
. 1999 Feb 28;354(1381):379–386. doi: 10.1098/rstb.1999.0390

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

M A Rahman 1, A C Ashton 1, F A Meunier 1, B A Davletov 1, J O Dolly 1, Y A Ushkaryov 1
PMCID: PMC1692485  PMID: 10212487

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.

Full Text

The Full Text of this article is available as a PDF (186.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barnett D. W., Liu J., Misler S. Single-cell measurements of quantal secretion induced by alpha-latrotoxin from rat adrenal chromaffin cells: dependence on extracellular Ca2+. Pflugers Arch. 1996 Oct;432(6):1039–1046. doi: 10.1007/s004240050232. [DOI] [PubMed] [Google Scholar]
  2. Bennett C. F., Mong S., Wu H. L., Clark M. A., Wheeler L., Crooke S. T. Inhibition of phosphoinositide-specific phospholipase C by manoalide. Mol Pharmacol. 1987 Nov;32(5):587–593. [PubMed] [Google Scholar]
  3. Blasi J., Chapman E. R., Yamasaki S., Binz T., Niemann H., Jahn R. Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin. EMBO J. 1993 Dec;12(12):4821–4828. doi: 10.1002/j.1460-2075.1993.tb06171.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Broadie K., Prokop A., Bellen H. J., O'Kane C. J., Schulze K. L., Sweeney S. T. Syntaxin and synaptobrevin function downstream of vesicle docking in Drosophila. Neuron. 1995 Sep;15(3):663–673. doi: 10.1016/0896-6273(95)90154-x. [DOI] [PubMed] [Google Scholar]
  5. Capogna M., Gähwiler B. H., Thompson S. M. Calcium-independent actions of alpha-latrotoxin on spontaneous and evoked synaptic transmission in the hippocampus. J Neurophysiol. 1996 Nov;76(5):3149–3158. doi: 10.1152/jn.1996.76.5.3149. [DOI] [PubMed] [Google Scholar]
  6. Carrasco M. A., Gaudry-Talarmain Y. M., Molgo J. Ca(2+)-dependent changes of acetylcholine release and IP3 mass in Torpedo cholinergic synaptosomes. Neurochem Int. 1997 Mar;30(3):321–327. doi: 10.1016/s0197-0186(96)00027-7. [DOI] [PubMed] [Google Scholar]
  7. Ceccarelli B., Hurlbut W. P. Ca2+-dependent recycling of synaptic vesicles at the frog neuromuscular junction. J Cell Biol. 1980 Oct;87(1):297–303. doi: 10.1083/jcb.87.1.297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Davletov B. A., Meunier F. A., Ashton A. C., Matsushita H., Hirst W. D., Lelianova V. G., Wilkin G. P., Dolly J. O., Ushkaryov Y. A. Vesicle exocytosis stimulated by alpha-latrotoxin is mediated by latrophilin and requires both external and stored Ca2+. EMBO J. 1998 Jul 15;17(14):3909–3920. doi: 10.1093/emboj/17.14.3909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davletov B. A., Shamotienko O. G., Lelianova V. G., Grishin E. V., Ushkaryov Y. A. Isolation and biochemical characterization of a Ca2+-independent alpha-latrotoxin-binding protein. J Biol Chem. 1996 Sep 20;271(38):23239–23245. doi: 10.1074/jbc.271.38.23239. [DOI] [PubMed] [Google Scholar]
  10. De Potter W. P., Partoens P., Schoups A., Llona I., Coen E. P. Noradrenergic neurons release both noradrenaline and neuropeptide Y from a single pool: the large dense cored vesicles. Synapse. 1997 Jan;25(1):44–55. doi: 10.1002/(SICI)1098-2396(199701)25:1<44::AID-SYN6>3.0.CO;2-F. [DOI] [PubMed] [Google Scholar]
  11. FINKELSTEIN A., Rubin L. L., Tzeng M. C. Black widow spider venom: effect of purified toxin on lipid bilayer membranes. Science. 1976 Sep 10;193(4257):1009–1011. doi: 10.1126/science.948756. [DOI] [PubMed] [Google Scholar]
  12. Finch E. A., Turner T. J., Goldin S. M. Calcium as a coagonist of inositol 1,4,5-trisphosphate-induced calcium release. Science. 1991 Apr 19;252(5004):443–446. doi: 10.1126/science.2017683. [DOI] [PubMed] [Google Scholar]
  13. Foran P., Lawrence G. W., Shone C. C., Foster K. A., Dolly J. O. Botulinum neurotoxin C1 cleaves both syntaxin and SNAP-25 in intact and permeabilized chromaffin cells: correlation with its blockade of catecholamine release. Biochemistry. 1996 Feb 27;35(8):2630–2636. doi: 10.1021/bi9519009. [DOI] [PubMed] [Google Scholar]
  14. Grasso A., Alemà S., Rufini S., Senni M. I. Black widow spider toxin-induced calcium fluxes and transmitter release in a neurosecretory cell line. Nature. 1980 Feb 21;283(5749):774–776. doi: 10.1038/283774a0. [DOI] [PubMed] [Google Scholar]
  15. Grasso A., Pelliccia M., Alemà S. Characterization of alpha-latrotoxin interaction with rat brain synaptosomes and PC12 cells. Toxicon. 1982;20(1):149–156. doi: 10.1016/0041-0101(82)90184-2. [DOI] [PubMed] [Google Scholar]
  16. Henkel A. W., Betz W. J. Monitoring of black widow spider venom (BWSV) induced exo- and endocytosis in living frog motor nerve terminals with FM1-43. Neuropharmacology. 1995 Nov;34(11):1397–1406. doi: 10.1016/0028-3908(95)00126-q. [DOI] [PubMed] [Google Scholar]
  17. Hurlbut W. P., Chieregatti E., Valtorta F., Haimann C. Alpha-latrotoxin channels in neuroblastoma cells. J Membr Biol. 1994 Feb;138(1):91–102. doi: 10.1007/BF00211072. [DOI] [PubMed] [Google Scholar]
  18. Jin W., Lo T. M., Loh H. H., Thayer S. A. U73122 inhibits phospholipase C-dependent calcium mobilization in neuronal cells. Brain Res. 1994 Apr 11;642(1-2):237–243. doi: 10.1016/0006-8993(94)90927-x. [DOI] [PubMed] [Google Scholar]
  19. Kozawa O., Hoshijima M., Tanimoto T., Ohmori T., Takai Y. Similar physical and kinetic properties of rat brain synaptic membrane and cytosol phosphoinositide phospholipases C. Biochem Biophys Res Commun. 1987 May 29;145(1):218–227. doi: 10.1016/0006-291x(87)91309-x. [DOI] [PubMed] [Google Scholar]
  20. Krasnoperov V. G., Bittner M. A., Beavis R., Kuang Y., Salnikow K. V., Chepurny O. G., Little A. R., Plotnikov A. N., Wu D., Holz R. W. alpha-Latrotoxin stimulates exocytosis by the interaction with a neuronal G-protein-coupled receptor. Neuron. 1997 Jun;18(6):925–937. doi: 10.1016/s0896-6273(00)80332-3. [DOI] [PubMed] [Google Scholar]
  21. Lang J., Ushkaryov Y., Grasso A., Wollheim C. B. Ca2+-independent insulin exocytosis induced by alpha-latrotoxin requires latrophilin, a G protein-coupled receptor. EMBO J. 1998 Feb 2;17(3):648–657. doi: 10.1093/emboj/17.3.648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lelianova V. G., Davletov B. A., Sterling A., Rahman M. A., Grishin E. V., Totty N. F., Ushkaryov Y. A. Alpha-latrotoxin receptor, latrophilin, is a novel member of the secretin family of G protein-coupled receptors. J Biol Chem. 1997 Aug 22;272(34):21504–21508. doi: 10.1074/jbc.272.34.21504. [DOI] [PubMed] [Google Scholar]
  23. Matteoli M., Haimann C., Torri-Tarelli F., Polak J. M., Ceccarelli B., De Camilli P. Differential effect of alpha-latrotoxin on exocytosis from small synaptic vesicles and from large dense-core vesicles containing calcitonin gene-related peptide at the frog neuromuscular junction. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7366–7370. doi: 10.1073/pnas.85.19.7366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McMahon H. T., Rosenthal L., Meldolesi J., Nicholls D. G. Alpha-latrotoxin releases both vesicular and cytoplasmic glutamate from isolated nerve terminals. J Neurochem. 1990 Dec;55(6):2039–2047. doi: 10.1111/j.1471-4159.1990.tb05793.x. [DOI] [PubMed] [Google Scholar]
  25. McPherson P. S., Kim Y. K., Valdivia H., Knudson C. M., Takekura H., Franzini-Armstrong C., Coronado R., Campbell K. P. The brain ryanodine receptor: a caffeine-sensitive calcium release channel. Neuron. 1991 Jul;7(1):17–25. doi: 10.1016/0896-6273(91)90070-g. [DOI] [PubMed] [Google Scholar]
  26. Meldolesi J., Huttner W. B., Tsien R. Y., Pozzan T. Free cytoplasmic Ca2+ and neurotransmitter release: studies on PC12 cells and synaptosomes exposed to alpha-latrotoxin. Proc Natl Acad Sci U S A. 1984 Jan;81(2):620–624. doi: 10.1073/pnas.81.2.620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Meldolesi J., Madeddu L., Torda M., Gatti G., Niutta E. The effect of alpha-latrotoxin on the neurosecretory PC12 cell line: studies on toxin binding and stimulation of transmitter release. Neuroscience. 1983 Nov;10(3):997–1009. doi: 10.1016/0306-4522(83)90238-5. [DOI] [PubMed] [Google Scholar]
  28. Meldolesi J. Studies on alpha-latrotoxin receptors in rat brain synaptosomes: correlation between toxin binding and stimulation of transmitter release. J Neurochem. 1982 Jun;38(6):1559–1569. doi: 10.1111/j.1471-4159.1982.tb06633.x. [DOI] [PubMed] [Google Scholar]
  29. Michelena P., de la Fuente M. T., Vega T., Lara B., López M. G., Gandía L., García A. G. Drastic facilitation by alpha-latrotoxin of bovine chromaffin cell exocytosis without measurable enhancement of Ca2+ entry or [Ca2+]i. J Physiol. 1997 Aug 1;502(Pt 3):481–496. doi: 10.1111/j.1469-7793.1997.481bj.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Misler S., Hurlbut W. P. Action of black widow spider venom on quantized release of acetylcholine at the frog neuromuscular junction: dependence upon external Mg2+. Proc Natl Acad Sci U S A. 1979 Feb;76(2):991–995. doi: 10.1073/pnas.76.2.991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Missler M., Südhof T. C. Neurexins: three genes and 1001 products. Trends Genet. 1998 Jan;14(1):20–26. doi: 10.1016/S0168-9525(97)01324-3. [DOI] [PubMed] [Google Scholar]
  32. Nicholls D. G., Rugolo M., Scott I. G., Meldolesi J. alpha-latrotoxin of black widow spider venom depolarizes the plasma membrane, induces massive calcium influx, and stimulates transmitter release in guinea pig brain synaptosomes. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7924–7928. doi: 10.1073/pnas.79.24.7924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rosenthal L., Meldolesi J. Alpha-latrotoxin and related toxins. Pharmacol Ther. 1989;42(1):115–134. doi: 10.1016/0163-7258(89)90024-7. [DOI] [PubMed] [Google Scholar]
  34. Rosenthal L., Zacchetti D., Madeddu L., Meldolesi J. Mode of action of alpha-latrotoxin: role of divalent cations in Ca2(+)-dependent and Ca2(+)-independent effects mediated by the toxin. Mol Pharmacol. 1990 Dec;38(6):917–923. [PubMed] [Google Scholar]
  35. Schiavo G., Poulain B., Rossetto O., Benfenati F., Tauc L., Montecucco C. Tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc. EMBO J. 1992 Oct;11(10):3577–3583. doi: 10.1002/j.1460-2075.1992.tb05441.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schiavo G., Rossetto O., Catsicas S., Polverino de Laureto P., DasGupta B. R., Benfenati F., Montecucco C. Identification of the nerve terminal targets of botulinum neurotoxin serotypes A, D, and E. J Biol Chem. 1993 Nov 15;268(32):23784–23787. [PubMed] [Google Scholar]
  37. Selbie L. A., Hill S. J. G protein-coupled-receptor cross-talk: the fine-tuning of multiple receptor-signalling pathways. Trends Pharmacol Sci. 1998 Mar;19(3):87–93. doi: 10.1016/s0165-6147(97)01166-8. [DOI] [PubMed] [Google Scholar]
  38. Smith R. J., Sam L. M., Justen J. M., Bundy G. L., Bala G. A., Bleasdale J. E. Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness. J Pharmacol Exp Ther. 1990 May;253(2):688–697. [PubMed] [Google Scholar]
  39. Söllner T., Bennett M. K., Whiteheart S. W., Scheller R. H., Rothman J. E. A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell. 1993 Nov 5;75(3):409–418. doi: 10.1016/0092-8674(93)90376-2. [DOI] [PubMed] [Google Scholar]
  40. Thastrup O., Cullen P. J., Drøbak B. K., Hanley M. R., Dawson A. P. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2466–2470. doi: 10.1073/pnas.87.7.2466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tse F. W., Tse A., Hille B., Horstmann H., Almers W. Local Ca2+ release from internal stores controls exocytosis in pituitary gonadotrophs. Neuron. 1997 Jan;18(1):121–132. doi: 10.1016/s0896-6273(01)80051-9. [DOI] [PubMed] [Google Scholar]
  42. Tzeng M. C., Siekevitz P. The binding interaction between alpha-latrotoxin from black widow spider venom and a dog cerebral cortex synaptosomal membrane preparation. J Neurochem. 1979 Jul;33(1):263–274. doi: 10.1111/j.1471-4159.1979.tb11728.x. [DOI] [PubMed] [Google Scholar]
  43. Ushkaryov Y. A., Petrenko A. G., Geppert M., Südhof T. C. Neurexins: synaptic cell surface proteins related to the alpha-latrotoxin receptor and laminin. Science. 1992 Jul 3;257(5066):50–56. doi: 10.1126/science.1621094. [DOI] [PubMed] [Google Scholar]
  44. Vicentini L. M., Meldolesi J. alpha Latrotoxin of black widow spider venom binds to a specific receptor coupled to phosphoinositide breakdown in PC12 cells. Biochem Biophys Res Commun. 1984 Jun 15;121(2):538–544. doi: 10.1016/0006-291x(84)90215-8. [DOI] [PubMed] [Google Scholar]

Articles from Philosophical Transactions of the Royal Society B: Biological Sciences are provided here courtesy of The Royal Society

RESOURCES