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. 1997 Mar 1;322(Pt 2):385–391. doi: 10.1042/bj3220385

Dominant negative mutant of ionotropic glutamate receptor subunit GluR3: implications for the role of a cysteine residue for its channel activity and pharmacological properties.

K Watase 1, M Sekiguchi 1, T A Matsui 1, Y Tagawa 1, K Wada 1
PMCID: PMC1218203  PMID: 9065754

Abstract

We reported that a 33-amino-acid deletion (from tyrosine-715 to glycine-747) in a putative extracellular loop of GluR3 produced a mutant that exhibited dominant negative effects upon the functional expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors [Sekiguchi et al. (1994) J. Biol. Chem. 269, 14559-14565]. In this study, we searched for a key residue in the dominant negative effects to explore the mechanism and examined the role of the residue in the function of the AMPA receptor. We prepared 20 GluR3 mutants with amino acid substitutions within the 33-amino-acid-region, and dominant negative effects were tested electrophysiologically in Xenopus oocytes co-expressing the mutant and normal subunits. Among the mutants, only a GluR3 mutant in which an original cysteine (Cys)-722 was replaced by alanine exhibited a dominant negative effect comparable with that of the original mutant in which the entire 33-amino-acid segment is deleted. The co-expression of the Cys-722 mutant did not inhibit the translation of normal subunits in oocytes. The Cys-722 mutant formed a functional homomeric receptor with significantly higher affinity for glutamate or kainate than a homomeric GluR3 receptor. The Cys-722 mutation greatly enhanced the sensitivity of GluR3 for aniracetam, which alters kinetic properties of AMPA receptors. The kainate-induced currents in oocytes expressing the Cys-722 mutant alone showed strong inward rectification. These results suggest that the Cys-722 in GluR3 is important for dominant negative effects and plays a crucial role in the determination of pharmacological properties in AMPA receptor function.

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Selected References

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  1. Ambros-Ingerson J., Lynch G. Channel gating kinetics and synaptic efficacy: a hypothesis for expression of long-term potentiation. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7903–7907. doi: 10.1073/pnas.90.16.7903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bennett J. A., Dingledine R. Topology profile for a glutamate receptor: three transmembrane domains and a channel-lining reentrant membrane loop. Neuron. 1995 Feb;14(2):373–384. doi: 10.1016/0896-6273(95)90293-7. [DOI] [PubMed] [Google Scholar]
  3. Blackstone C. D., Moss S. J., Martin L. J., Levey A. I., Price D. L., Huganir R. L. Biochemical characterization and localization of a non-N-methyl-D-aspartate glutamate receptor in rat brain. J Neurochem. 1992 Mar;58(3):1118–1126. doi: 10.1111/j.1471-4159.1992.tb09370.x. [DOI] [PubMed] [Google Scholar]
  4. Boulter J., Hollmann M., O'Shea-Greenfield A., Hartley M., Deneris E., Maron C., Heinemann S. Molecular cloning and functional expression of glutamate receptor subunit genes. Science. 1990 Aug 31;249(4972):1033–1037. doi: 10.1126/science.2168579. [DOI] [PubMed] [Google Scholar]
  5. Ferrer-Montiel A. V., Montal M. Pentameric subunit stoichiometry of a neuronal glutamate receptor. Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2741–2744. doi: 10.1073/pnas.93.7.2741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hollmann M., Heinemann S. Cloned glutamate receptors. Annu Rev Neurosci. 1994;17:31–108. doi: 10.1146/annurev.ne.17.030194.000335. [DOI] [PubMed] [Google Scholar]
  7. Hollmann M., Maron C., Heinemann S. N-glycosylation site tagging suggests a three transmembrane domain topology for the glutamate receptor GluR1. Neuron. 1994 Dec;13(6):1331–1343. doi: 10.1016/0896-6273(94)90419-7. [DOI] [PubMed] [Google Scholar]
  8. Hurtley S. M., Helenius A. Protein oligomerization in the endoplasmic reticulum. Annu Rev Cell Biol. 1989;5:277–307. doi: 10.1146/annurev.cb.05.110189.001425. [DOI] [PubMed] [Google Scholar]
  9. Isaacson J. S., Nicoll R. A. Aniracetam reduces glutamate receptor desensitization and slows the decay of fast excitatory synaptic currents in the hippocampus. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10936–10940. doi: 10.1073/pnas.88.23.10936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lomeli H., Mosbacher J., Melcher T., Höger T., Geiger J. R., Kuner T., Monyer H., Higuchi M., Bach A., Seeburg P. H. Control of kinetic properties of AMPA receptor channels by nuclear RNA editing. Science. 1994 Dec 9;266(5191):1709–1713. doi: 10.1126/science.7992055. [DOI] [PubMed] [Google Scholar]
  11. Matsui T., Sekiguchi M., Hashimoto A., Tomita U., Nishikawa T., Wada K. Functional comparison of D-serine and glycine in rodents: the effect on cloned NMDA receptors and the extracellular concentration. J Neurochem. 1995 Jul;65(1):454–458. doi: 10.1046/j.1471-4159.1995.65010454.x. [DOI] [PubMed] [Google Scholar]
  12. McIlhinney R. A., Molnár E. Characterization, cell-surface expression and ligand-binding properties of different truncated N-terminal extracellular domains of the ionotropic glutamate receptor subunit GluR1. Biochem J. 1996 Apr 1;315(Pt 1):217–225. doi: 10.1042/bj3150217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mosbacher J., Schoepfer R., Monyer H., Burnashev N., Seeburg P. H., Ruppersberg J. P. A molecular determinant for submillisecond desensitization in glutamate receptors. Science. 1994 Nov 11;266(5187):1059–1062. doi: 10.1126/science.7973663. [DOI] [PubMed] [Google Scholar]
  14. Nakanishi S. Molecular diversity of glutamate receptors and implications for brain function. Science. 1992 Oct 23;258(5082):597–603. doi: 10.1126/science.1329206. [DOI] [PubMed] [Google Scholar]
  15. Nakazawa K., Mikawa S., Hashikawa T., Ito M. Transient and persistent phosphorylation of AMPA-type glutamate receptor subunits in cerebellar Purkinje cells. Neuron. 1995 Sep;15(3):697–709. doi: 10.1016/0896-6273(95)90157-4. [DOI] [PubMed] [Google Scholar]
  16. Partin K. M., Bowie D., Mayer M. L. Structural determinants of allosteric regulation in alternatively spliced AMPA receptors. Neuron. 1995 Apr;14(4):833–843. doi: 10.1016/0896-6273(95)90227-9. [DOI] [PubMed] [Google Scholar]
  17. Patneau D. K., Mayer M. L. Kinetic analysis of interactions between kainate and AMPA: evidence for activation of a single receptor in mouse hippocampal neurons. Neuron. 1991 May;6(5):785–798. doi: 10.1016/0896-6273(91)90175-y. [DOI] [PubMed] [Google Scholar]
  18. Seeburg P. H. The TINS/TiPS Lecture. The molecular biology of mammalian glutamate receptor channels. Trends Neurosci. 1993 Sep;16(9):359–365. doi: 10.1016/0166-2236(93)90093-2. [DOI] [PubMed] [Google Scholar]
  19. Sekiguchi M., Doi K., Zhu W. S., Watase K., Yokotani N., Wada K., Wenthold R. J. A deletion in the second cytoplasmic loop of GluR3 produces a dominant negative mutant of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor. J Biol Chem. 1994 May 20;269(20):14559–14565. [PubMed] [Google Scholar]
  20. Sommer B., Keinänen K., Verdoorn T. A., Wisden W., Burnashev N., Herb A., Köhler M., Takagi T., Sakmann B., Seeburg P. H. Flip and flop: a cell-specific functional switch in glutamate-operated channels of the CNS. Science. 1990 Sep 28;249(4976):1580–1585. doi: 10.1126/science.1699275. [DOI] [PubMed] [Google Scholar]
  21. Stern-Bach Y., Bettler B., Hartley M., Sheppard P. O., O'Hara P. J., Heinemann S. F. Agonist selectivity of glutamate receptors is specified by two domains structurally related to bacterial amino acid-binding proteins. Neuron. 1994 Dec;13(6):1345–1357. doi: 10.1016/0896-6273(94)90420-0. [DOI] [PubMed] [Google Scholar]
  22. Sullivan J. M., Traynelis S. F., Chen H. S., Escobar W., Heinemann S. F., Lipton S. A. Identification of two cysteine residues that are required for redox modulation of the NMDA subtype of glutamate receptor. Neuron. 1994 Oct;13(4):929–936. doi: 10.1016/0896-6273(94)90258-5. [DOI] [PubMed] [Google Scholar]
  23. Trussell L. O., Fischbach G. D. Glutamate receptor desensitization and its role in synaptic transmission. Neuron. 1989 Aug;3(2):209–218. doi: 10.1016/0896-6273(89)90034-2. [DOI] [PubMed] [Google Scholar]
  24. Vyklicky L., Jr, Patneau D. K., Mayer M. L. Modulation of excitatory synaptic transmission by drugs that reduce desensitization at AMPA/kainate receptors. Neuron. 1991 Dec;7(6):971–984. doi: 10.1016/0896-6273(91)90342-w. [DOI] [PubMed] [Google Scholar]
  25. Wenthold R. J., Yokotani N., Doi K., Wada K. Immunochemical characterization of the non-NMDA glutamate receptor using subunit-specific antibodies. Evidence for a hetero-oligomeric structure in rat brain. J Biol Chem. 1992 Jan 5;267(1):501–507. [PubMed] [Google Scholar]

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