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. 1999 Oct 15;343(Pt 2):413–418.

Inhibition of glutamate uptake by a polypeptide toxin (phoneutriatoxin 3-4) from the spider Phoneutria nigriventer.

H J Reis 1, M A Prado 1, E Kalapothakis 1, M N Cordeiro 1, C R Diniz 1, L A De Marco 1, M V Gomez 1, M A Romano-Silva 1
PMCID: PMC1220569  PMID: 10510308

Abstract

Glutamate concentration increases significantly in the extracellular compartment during brain ischaemia and anoxia. This increase has an important Ca(2+)-independent component, which is due in part to the reversal of glutamate transporters of the plasma membrane of neurons and glia. The toxin phoneutriatoxin 3-4 (Tx3-4) from the spider Phoneutria nigriventer has been reported to decrease the evoked glutamate release from synaptosomes by inhibiting Ca(2+) entry via voltage-dependent Ca(2+) channels. However, we report here that Tx3-4 is also able to inhibit the uptake of glutamate by synaptosomes in a time-dependent manner and that this inhibition in turn leads to a decrease in the Ca(2+)-independent release of glutamate. No other polypeptide toxin so far described has this effect. Our results suggest that Tx3-4 can be a valuable tool in the investigation of function and dysfunction of glutamatergic neurotransmission in diseases such as ischaemia.

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Selected References

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  1. Araújo D. A., Cordeiro M. N., Diniz C. R., Beirão P. S. Effects of a toxic fraction, PhTx2, from the spider Phoneutria nigriventer on the sodium current. Naunyn Schmiedebergs Arch Pharmacol. 1993 Feb;347(2):205–208. doi: 10.1007/BF00169268. [DOI] [PubMed] [Google Scholar]
  2. Arriza J. L., Eliasof S., Kavanaugh M. P., Amara S. G. Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):4155–4160. doi: 10.1073/pnas.94.8.4155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arriza J. L., Fairman W. A., Wadiche J. I., Murdoch G. H., Kavanaugh M. P., Amara S. G. Functional comparisons of three glutamate transporter subtypes cloned from human motor cortex. J Neurosci. 1994 Sep;14(9):5559–5569. doi: 10.1523/JNEUROSCI.14-09-05559.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Attwell D., Barbour B., Szatkowski M. Nonvesicular release of neurotransmitter. Neuron. 1993 Sep;11(3):401–407. doi: 10.1016/0896-6273(93)90145-h. [DOI] [PubMed] [Google Scholar]
  5. Benveniste H., Drejer J., Schousboe A., Diemer N. H. Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J Neurochem. 1984 Nov;43(5):1369–1374. doi: 10.1111/j.1471-4159.1984.tb05396.x. [DOI] [PubMed] [Google Scholar]
  6. Bergles D. E., Dzubay J. A., Jahr C. E. Glutamate transporter currents in bergmann glial cells follow the time course of extrasynaptic glutamate. Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14821–14825. doi: 10.1073/pnas.94.26.14821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cassola A. C., Jaffe H., Fales H. M., Afeche S. C., Magnoli F., Cipolla-Neto J. ω-Phonetoxin-IIA: a calcium channel blocker from the spider Phoneutria nigriventer. Pflugers Arch. 1998 Jul;436(4):545–552. doi: 10.1007/s004240050670. [DOI] [PubMed] [Google Scholar]
  8. Choi D. W., Maulucci-Gedde M., Kriegstein A. R. Glutamate neurotoxicity in cortical cell culture. J Neurosci. 1987 Feb;7(2):357–368. doi: 10.1523/JNEUROSCI.07-02-00357.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cordeiro M. do N., de Figueiredo S. G., Valentim A. do C., Diniz C. R., von Eickstedt V. R., Gilroy J., Richardson M. Purification and amino acid sequences of six Tx3 type neurotoxins from the venom of the Brazilian 'armed' spider Phoneutria nigriventer (Keys). Toxicon. 1993 Jan;31(1):35–42. doi: 10.1016/0041-0101(93)90354-l. [DOI] [PubMed] [Google Scholar]
  10. Dixon J. F., Hokin L. E. Lithium acutely inhibits and chronically up-regulates and stabilizes glutamate uptake by presynaptic nerve endings in mouse cerebral cortex. Proc Natl Acad Sci U S A. 1998 Jul 7;95(14):8363–8368. doi: 10.1073/pnas.95.14.8363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dowd L. A., Coyle A. J., Rothstein J. D., Pritchett D. B., Robinson M. B. Comparison of Na+-dependent glutamate transport activity in synaptosomes, C6 glioma, and Xenopus oocytes expressing excitatory amino acid carrier 1 (EAAC1). Mol Pharmacol. 1996 Mar;49(3):465–473. [PubMed] [Google Scholar]
  12. Dunkley P. R., Heath J. W., Harrison S. M., Jarvie P. E., Glenfield P. J., Rostas J. A. A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: homogeneity and morphology of subcellular fractions. Brain Res. 1988 Feb 16;441(1-2):59–71. doi: 10.1016/0006-8993(88)91383-2. [DOI] [PubMed] [Google Scholar]
  13. Ferkany J., Coyle J. T. Heterogeneity of sodium-dependent excitatory amino acid uptake mechanisms in rat brain. J Neurosci Res. 1986;16(3):491–503. doi: 10.1002/jnr.490160305. [DOI] [PubMed] [Google Scholar]
  14. Garlin A. B., Sinor A. D., Sinor J. D., Jee S. H., Grinspan J. B., Robinson M. B. Pharmacology of sodium-dependent high-affinity L-[3H]glutamate transport in glial cultures. J Neurochem. 1995 Jun;64(6):2572–2580. doi: 10.1046/j.1471-4159.1995.64062572.x. [DOI] [PubMed] [Google Scholar]
  15. Guatimosim C., Romano-Silva M. A., Cruz J. S., Beirão P. S., Kalapothakis E., Moraes-Santos T., Cordeiro M. N., Diniz C. R., Gomez M. V., Prado M. A. A toxin from the spider Phoneutria nigriventer that blocks calcium channels coupled to exocytosis. Br J Pharmacol. 1997 Oct;122(3):591–597. doi: 10.1038/sj.bjp.0701381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Henriques M. C., Gomez M. V. Effect of scorpion venom, tityustoxin, on the uptake of calcium by isolated nerve ending particles from brain. Brain Res. 1975 Jul 25;93(1):182–187. doi: 10.1016/0006-8993(75)90299-1. [DOI] [PubMed] [Google Scholar]
  17. Jacques Y., Fosset M., Lazdunski M. Molecular properties of the action potential Na+ ionophore in neuroblastoma cells. Interactions with neurotoxins. J Biol Chem. 1978 Oct 25;253(20):7383–7392. [PubMed] [Google Scholar]
  18. Kanai Y., Hediger M. A. Primary structure and functional characterization of a high-affinity glutamate transporter. Nature. 1992 Dec 3;360(6403):467–471. doi: 10.1038/360467a0. [DOI] [PubMed] [Google Scholar]
  19. Kanai Y., Smith C. P., Hediger M. A. The elusive transporters with a high affinity for glutamate. Trends Neurosci. 1993 Sep;16(9):365–370. doi: 10.1016/0166-2236(93)90094-3. [DOI] [PubMed] [Google Scholar]
  20. Kauppinen R. A., McMahon H. T., Nicholls D. G. Ca2+-dependent and Ca2+-independent glutamate release, energy status and cytosolic free Ca2+ concentration in isolated nerve terminals following metabolic inhibition: possible relevance to hypoglycaemia and anoxia. Neuroscience. 1988 Oct;27(1):175–182. doi: 10.1016/0306-4522(88)90228-x. [DOI] [PubMed] [Google Scholar]
  21. Kimelberg H. K., Rutledge E., Goderie S., Charniga C. Astrocytic swelling due to hypotonic or high K+ medium causes inhibition of glutamate and aspartate uptake and increases their release. J Cereb Blood Flow Metab. 1995 May;15(3):409–416. doi: 10.1038/jcbfm.1995.51. [DOI] [PubMed] [Google Scholar]
  22. Kushmerick C., Kalapothakis E., Beirão P. S., Penaforte C. L., Prado V. F., Cruz J. S., Diniz C. R., Cordeiro M. N., Gomez M. V., Romano-Silva M. A. Phoneutria nigriventer toxin Tx3-1 blocks A-type K+ currents controlling Ca2+ oscillation frequency in GH3 cells. J Neurochem. 1999 Apr;72(4):1472–1481. doi: 10.1046/j.1471-4159.1999.721472.x. [DOI] [PubMed] [Google Scholar]
  23. Levy L. M., Warr O., Attwell D. Stoichiometry of the glial glutamate transporter GLT-1 expressed inducibly in a Chinese hamster ovary cell line selected for low endogenous Na+-dependent glutamate uptake. J Neurosci. 1998 Dec 1;18(23):9620–9628. doi: 10.1523/JNEUROSCI.18-23-09620.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Miranda D. M., Romano-Silva M. A., Kalapothakis E., Diniz C. R., Cordeiro M. N., Santos T. M., Prado M. A., Gomez M. V. Phoneutria nigriventer toxins block tityustoxin-induced calcium influx in synaptosomes. Neuroreport. 1998 May 11;9(7):1371–1373. doi: 10.1097/00001756-199805110-00022. [DOI] [PubMed] [Google Scholar]
  25. Nakamura Y., Iga K., Shibata T., Shudo M., Kataoka K. Glial plasmalemmal vesicles: a subcellular fraction from rat hippocampal homogenate distinct from synaptosomes. Glia. 1993 Sep;9(1):48–56. doi: 10.1002/glia.440090107. [DOI] [PubMed] [Google Scholar]
  26. Nakamura Y., Kubo H., Kataoka K. Uptake of transmitter amino acids by glial plasmalemmal vesicles from different regions of rat central nervous system. Neurochem Res. 1994 Sep;19(9):1145–1150. doi: 10.1007/BF00965148. [DOI] [PubMed] [Google Scholar]
  27. Neal M. J., Cunningham J. R., Hutson P. H., Hogg J. Effects of ischaemia on neurotransmitter release from the isolated retina. J Neurochem. 1994 Mar;62(3):1025–1033. doi: 10.1046/j.1471-4159.1994.62031025.x. [DOI] [PubMed] [Google Scholar]
  28. Nicholls D. G., Sihra T. S., Sanchez-Prieto J. Calcium-dependent and -independent release of glutamate from synaptosomes monitored by continuous fluorometry. J Neurochem. 1987 Jul;49(1):50–57. doi: 10.1111/j.1471-4159.1987.tb03393.x. [DOI] [PubMed] [Google Scholar]
  29. Pan-Hou H., Suda Y., Sumi M., Yoshioka M., Kawai N. A spider toxin (JSTX) inhibits L-glutamate uptake by rat brain synaptosomes. Brain Res. 1989 Jan 9;476(2):354–357. doi: 10.1016/0006-8993(89)91258-4. [DOI] [PubMed] [Google Scholar]
  30. Pines G., Danbolt N. C., Bjørås M., Zhang Y., Bendahan A., Eide L., Koepsell H., Storm-Mathisen J., Seeberg E., Kanner B. I. Cloning and expression of a rat brain L-glutamate transporter. Nature. 1992 Dec 3;360(6403):464–467. doi: 10.1038/360464a0. [DOI] [PubMed] [Google Scholar]
  31. Plakidou-Dymock S., McGivan J. D. Regulation of the glutamate transporter by amino acid deprivation and associated effects on the level of EAAC1 mRNA in the renal epithelial cell line NBL-I. Biochem J. 1993 Nov 1;295(Pt 3):749–755. doi: 10.1042/bj2950749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Prado M. A., Guatimosim C., Gomez M. V., Diniz C. R., Cordeiro M. N., Romano-Silva M. A. A novel tool for the investigation of glutamate release from rat cerebrocortical synaptosomes: the toxin Tx3-3 from the venom of the spider Phoneutria nigriventer. Biochem J. 1996 Feb 15;314(Pt 1):145–150. doi: 10.1042/bj3140145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rego A. C., Santos M. S., Oliveira C. R. Oxidative stress, hypoxia, and ischemia-like conditions increase the release of endogenous amino acids by distinct mechanisms in cultured retinal cells. J Neurochem. 1996 Jun;66(6):2506–2516. doi: 10.1046/j.1471-4159.1996.66062506.x. [DOI] [PubMed] [Google Scholar]
  34. Robinson M. B., Dowd L. A. Heterogeneity and functional properties of subtypes of sodium-dependent glutamate transporters in the mammalian central nervous system. Adv Pharmacol. 1997;37:69–115. doi: 10.1016/s1054-3589(08)60948-5. [DOI] [PubMed] [Google Scholar]
  35. Robinson M. B., Hunter-Ensor M., Sinor J. Pharmacologically distinct sodium-dependent L-[3H]glutamate transport processes in rat brain. Brain Res. 1991 Mar 29;544(2):196–202. doi: 10.1016/0006-8993(91)90054-y. [DOI] [PubMed] [Google Scholar]
  36. Romano-Silva M. A., Gomez M. V., Brammer M. J. Modulation of Ca(2+)-stimulated glutamate release from synaptosomes by Na+ entry through tetrodotoxin-sensitive channels. Biochem J. 1994 Dec 1;304(Pt 2):353–357. doi: 10.1042/bj3040353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Romano-Silva M. A., Ribeiro-Santos R., Ribeiro A. M., Gomez M. V., Diniz C. R., Cordeiro M. N., Brammer M. J. Rat cortical synaptosomes have more than one mechanism for Ca2+ entry linked to rapid glutamate release: studies using the Phoneutria nigriventer toxin PhTX2 and potassium depolarization. Biochem J. 1993 Dec 1;296(Pt 2):313–319. doi: 10.1042/bj2960313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rothstein J. D., Martin L., Levey A. I., Dykes-Hoberg M., Jin L., Wu D., Nash N., Kuncl R. W. Localization of neuronal and glial glutamate transporters. Neuron. 1994 Sep;13(3):713–725. doi: 10.1016/0896-6273(94)90038-8. [DOI] [PubMed] [Google Scholar]
  39. Rutledge E. M., Aschner M., Kimelberg H. K. Pharmacological characterization of swelling-induced D-[3H]aspartate release from primary astrocyte cultures. Am J Physiol. 1998 Jun;274(6 Pt 1):C1511–C1520. doi: 10.1152/ajpcell.1998.274.6.C1511. [DOI] [PubMed] [Google Scholar]
  40. Rutledge E. M., Kimelberg H. K. Release of [3H]-D-aspartate from primary astrocyte cultures in response to raised external potassium. J Neurosci. 1996 Dec 15;16(24):7803–7811. doi: 10.1523/JNEUROSCI.16-24-07803.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sanchez-Prieto J., Sihra T. S., Nicholls D. G. Characterization of the exocytotic release of glutamate from guinea-pig cerebral cortical synaptosomes. J Neurochem. 1987 Jul;49(1):58–64. doi: 10.1111/j.1471-4159.1987.tb03394.x. [DOI] [PubMed] [Google Scholar]
  42. Storck T., Schulte S., Hofmann K., Stoffel W. Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10955–10959. doi: 10.1073/pnas.89.22.10955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Turner T. J., Dunlap K. Pharmacological characterization of presynaptic calcium channels using subsecond biochemical measurements of synaptosomal neurosecretion. Neuropharmacology. 1995 Nov;34(11):1469–1478. doi: 10.1016/0028-3908(95)00133-q. [DOI] [PubMed] [Google Scholar]
  44. Wadiche J. I., Arriza J. L., Amara S. G., Kavanaugh M. P. Kinetics of a human glutamate transporter. Neuron. 1995 May;14(5):1019–1027. doi: 10.1016/0896-6273(95)90340-2. [DOI] [PubMed] [Google Scholar]
  45. Wang G. J., Chung H. J., Schnuer J., Pratt K., Zable A. C., Kavanaugh M. P., Rosenberg P. A. High affinity glutamate transport in rat cortical neurons in culture. Mol Pharmacol. 1998 Jan;53(1):88–96. doi: 10.1124/mol.53.1.88. [DOI] [PubMed] [Google Scholar]
  46. Watase K., Hashimoto K., Kano M., Yamada K., Watanabe M., Inoue Y., Okuyama S., Sakagawa T., Ogawa S., Kawashima N. Motor discoordination and increased susceptibility to cerebellar injury in GLAST mutant mice. Eur J Neurosci. 1998 Mar;10(3):976–988. doi: 10.1046/j.1460-9568.1998.00108.x. [DOI] [PubMed] [Google Scholar]

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