Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 2000 Nov;79(5):2454–2462. doi: 10.1016/S0006-3495(00)76488-5

NMDA channel gating is influenced by a tryptophan residue in the M2 domain but calcium permeation is not altered.

D P Buck 1, S M Howitt 1, J D Clements 1
PMCID: PMC1301130  PMID: 11053122

Abstract

N-Methyl-D-aspartate (NMDA) receptors are susceptible to open-channel block by dizolcipine (MK-801), ketamine and Mg(2+) and are permeable to Ca(2+). It is thought that a tryptophan residue in the second membrane-associated domain (M2) may form part of the binding site for open-channel blockers and contribute to Ca(2+) permeability. We tested this hypothesis using recombinant wild-type and mutant NMDA receptors expressed in HEK-293 cells. The tryptophan was mutated to a leucine (W-5L) in both the NMDAR1 and NMDAR2A subunits. MK-801 and ketamine progressively inhibited currents evoked by glutamate, and the rate of inhibition was increased by the W-5L mutation. An increase in open channel probability accounted for the acceleration. Fluctuation analysis of the glutamate-evoked current revealed that the NMDAR1 W-5L mutation increased channel mean open time, providing further evidence for an alteration in gating. However, the equilibrium affinities of Mg(2+) and ketamine were largely unaffected by the W-5L mutation, and Ca(2+) permeability was not decreased. Therefore, the M2 tryptophan residue of the NMDA channel is not involved in Ca(2+) permeation or the binding of open-channel blockers, but plays an important role in channel gating.

Full Text

The Full Text of this article is available as a PDF (137.0 KB).

Selected References

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

  1. Ascher P., Nowak L. The role of divalent cations in the N-methyl-D-aspartate responses of mouse central neurones in culture. J Physiol. 1988 May;399:247–266. doi: 10.1113/jphysiol.1988.sp017078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beck C., Wollmuth L. P., Seeburg P. H., Sakmann B., Kuner T. NMDAR channel segments forming the extracellular vestibule inferred from the accessibility of substituted cysteines. Neuron. 1999 Mar;22(3):559–570. doi: 10.1016/s0896-6273(00)80710-2. [DOI] [PubMed] [Google Scholar]
  3. Benveniste M., Mayer M. L. Trapping of glutamate and glycine during open channel block of rat hippocampal neuron NMDA receptors by 9-aminoacridine. J Physiol. 1995 Mar 1;483(Pt 2):367–384. doi: 10.1113/jphysiol.1995.sp020591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burnashev N., Monyer H., Seeburg P. H., Sakmann B. Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit. Neuron. 1992 Jan;8(1):189–198. doi: 10.1016/0896-6273(92)90120-3. [DOI] [PubMed] [Google Scholar]
  5. Burnashev N., Schoepfer R., Monyer H., Ruppersberg J. P., Günther W., Seeburg P. H., Sakmann B. Control by asparagine residues of calcium permeability and magnesium blockade in the NMDA receptor. Science. 1992 Sep 4;257(5075):1415–1419. doi: 10.1126/science.1382314. [DOI] [PubMed] [Google Scholar]
  6. Collingridge G. L., Lester R. A. Excitatory amino acid receptors in the vertebrate central nervous system. Pharmacol Rev. 1989 Jun;41(2):143–210. [PubMed] [Google Scholar]
  7. Dingledine R., Hume R. I., Heinemann S. F. Structural determinants of barium permeation and rectification in non-NMDA glutamate receptor channels. J Neurosci. 1992 Oct;12(10):4080–4087. doi: 10.1523/JNEUROSCI.12-10-04080.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ferrer-Montiel A. V., Sun W., Montal M. A single tryptophan on M2 of glutamate receptor channels confers high permeability to divalent cations. Biophys J. 1996 Aug;71(2):749–758. doi: 10.1016/S0006-3495(96)79274-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Huettner J. E., Bean B. P. Block of N-methyl-D-aspartate-activated current by the anticonvulsant MK-801: selective binding to open channels. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1307–1311. doi: 10.1073/pnas.85.4.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Iino M., Ozawa S., Tsuzuki K. Permeation of calcium through excitatory amino acid receptor channels in cultured rat hippocampal neurones. J Physiol. 1990 May;424:151–165. doi: 10.1113/jphysiol.1990.sp018060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jonas P., Burnashev N. Molecular mechanisms controlling calcium entry through AMPA-type glutamate receptor channels. Neuron. 1995 Nov;15(5):987–990. doi: 10.1016/0896-6273(95)90087-x. [DOI] [PubMed] [Google Scholar]
  12. Kuner T., Wollmuth L. P., Karlin A., Seeburg P. H., Sakmann B. Structure of the NMDA receptor channel M2 segment inferred from the accessibility of substituted cysteines. Neuron. 1996 Aug;17(2):343–352. doi: 10.1016/s0896-6273(00)80165-8. [DOI] [PubMed] [Google Scholar]
  13. Kutsuwada T., Kashiwabuchi N., Mori H., Sakimura K., Kushiya E., Araki K., Meguro H., Masaki H., Kumanishi T., Arakawa M. Molecular diversity of the NMDA receptor channel. Nature. 1992 Jul 2;358(6381):36–41. doi: 10.1038/358036a0. [DOI] [PubMed] [Google Scholar]
  14. Lester R. A., Clements J. D., Westbrook G. L., Jahr C. E. Channel kinetics determine the time course of NMDA receptor-mediated synaptic currents. Nature. 1990 Aug 9;346(6284):565–567. doi: 10.1038/346565a0. [DOI] [PubMed] [Google Scholar]
  15. Mayer M. L., Westbrook G. L., Guthrie P. B. Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones. Nature. 1984 May 17;309(5965):261–263. doi: 10.1038/309261a0. [DOI] [PubMed] [Google Scholar]
  16. Mayer M. L., Westbrook G. L. Permeation and block of N-methyl-D-aspartic acid receptor channels by divalent cations in mouse cultured central neurones. J Physiol. 1987 Dec;394:501–527. doi: 10.1113/jphysiol.1987.sp016883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mayer M. L., Westbrook G. L., Vyklický L., Jr Sites of antagonist action on N-methyl-D-aspartic acid receptors studied using fluctuation analysis and a rapid perfusion technique. J Neurophysiol. 1988 Aug;60(2):645–663. doi: 10.1152/jn.1988.60.2.645. [DOI] [PubMed] [Google Scholar]
  18. Mori H., Masaki H., Yamakura T., Mishina M. Identification by mutagenesis of a Mg(2+)-block site of the NMDA receptor channel. Nature. 1992 Aug 20;358(6388):673–675. doi: 10.1038/358673a0. [DOI] [PubMed] [Google Scholar]
  19. Moriyoshi K., Masu M., Ishii T., Shigemoto R., Mizuno N., Nakanishi S. Molecular cloning and characterization of the rat NMDA receptor. Nature. 1991 Nov 7;354(6348):31–37. doi: 10.1038/354031a0. [DOI] [PubMed] [Google Scholar]
  20. Premkumar L. S., Auerbach A. Identification of a high affinity divalent cation binding site near the entrance of the NMDA receptor channel. Neuron. 1996 Apr;16(4):869–880. doi: 10.1016/s0896-6273(00)80107-5. [DOI] [PubMed] [Google Scholar]
  21. Rosenmund C., Clements J. D., Westbrook G. L. Nonuniform probability of glutamate release at a hippocampal synapse. Science. 1993 Oct 29;262(5134):754–757. doi: 10.1126/science.7901909. [DOI] [PubMed] [Google Scholar]
  22. Schneggenburger R., Ascher P. Coupling of permeation and gating in an NMDA-channel pore mutant. Neuron. 1997 Jan;18(1):167–177. doi: 10.1016/s0896-6273(01)80055-6. [DOI] [PubMed] [Google Scholar]
  23. Schneggenburger R., Zhou Z., Konnerth A., Neher E. Fractional contribution of calcium to the cation current through glutamate receptor channels. Neuron. 1993 Jul;11(1):133–143. doi: 10.1016/0896-6273(93)90277-x. [DOI] [PubMed] [Google Scholar]
  24. Sharma G., Stevens C. F. Interactions between two divalent ion binding sites in N-methyl-D-aspartate receptor channels. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):14170–14175. doi: 10.1073/pnas.93.24.14170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Stern P., Cik M., Colquhoun D., Stephenson F. A. Single channel properties of cloned NMDA receptors in a human cell line: comparison with results from Xenopus oocytes. J Physiol. 1994 May 1;476(3):391–397. doi: 10.1113/jphysiol.1994.sp020140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Williams K., Pahk A. J., Kashiwagi K., Masuko T., Nguyen N. D., Igarashi K. The selectivity filter of the N-methyl-D-aspartate receptor: a tryptophan residue controls block and permeation of Mg2+. Mol Pharmacol. 1998 May;53(5):933–941. [PubMed] [Google Scholar]
  27. Wollmuth L. P., Sakmann B. Different mechanisms of Ca2+ transport in NMDA and Ca2+-permeable AMPA glutamate receptor channels. J Gen Physiol. 1998 Nov;112(5):623–636. doi: 10.1085/jgp.112.5.623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zarei M. M., Dani J. A. Ionic permeability characteristics of the N-methyl-D-aspartate receptor channel. J Gen Physiol. 1994 Feb;103(2):231–248. doi: 10.1085/jgp.103.2.231. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES