Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

The structure–energy landscape of NMDA receptor gating

Nature Chemical Biology volume 13, pages 1232–1238 (2017)Cite this article

Subjects

Abstract

N-Methyl-D-aspartate (NMDA) receptors are the main calcium-permeable excitatory receptors in the mammalian central nervous system. The NMDA receptor gating is complex, exhibiting multiple closed, open, and desensitized states; however, central questions regarding the conformations and energetics of the transmembrane domains as they relate to the gating states are still unanswered. Here, using single-molecule Förster resonance energy transfer (smFRET), we map the energy landscape of the first transmembrane segment of the Rattus norvegicus NMDA receptor under resting and various liganded conditions. These results show kinetically and structurally distinct changes associated with apo, agonist-bound, and inhibited receptors linked by a linear mechanism of gating at this site. Furthermore, the smFRET data suggest that allosteric inhibition by zinc occurs by an uncoupling of the agonist-induced changes at the extracellular domains from the gating motions leading to an apo-like state, while dizocilpine, a pore blocker, stabilizes multiple closely packed transmembrane states.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: smFRET constructs and characterization.
Figure 2: Denoised smFRET histograms of the NMDA receptor.
Figure 3: Dynamics of the NMDA receptor show differences in transitional behavior under different ligand conditions.
Figure 4: Transition maps and free energy diagrams of the NMDA receptor smFRET data.

Similar content being viewed by others

References

  1. Zhang, W., Howe, J.R. & Popescu, G.K. Distinct gating modes determine the biphasic relaxation of NMDA receptor currents. Nat. Neurosci. 11, 1373–1375 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Lape, R., Colquhoun, D. & Sivilotti, L.G. On the nature of partial agonism in the nicotinic receptor superfamily. Nature 454, 722–727 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Zhang, W., Devi, S.P., Tomita, S. & Howe, J.R. Auxiliary proteins promote modal gating of AMPA- and kainate-type glutamate receptors. Eur. J. Neurosci. 39, 1138–1147 (2014).

    PubMed  PubMed Central  Google Scholar 

  4. Tajima, N. et al. Activation of NMDA receptors and the mechanism of inhibition by ifenprodil. Nature 534, 63–68 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Popescu, G., Robert, A., Howe, J.R. & Auerbach, A. Reaction mechanism determines NMDA receptor response to repetitive stimulation. Nature 430, 790–793 (2004).

    CAS  PubMed  Google Scholar 

  6. Banke, T.G. & Traynelis, S.F. Activation of NR1/NR2B NMDA receptors. Nat. Neurosci. 6, 144–152 (2003).

    CAS  PubMed  Google Scholar 

  7. Kussius, C.L. & Popescu, G.K. Kinetic basis of partial agonism at NMDA receptors. Nat. Neurosci. 12, 1114–1120 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Zhu, S. et al. Mechanism of NMDA receptor inhibition and activation. Cell 165, 704–714 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Karakas, E. & Furukawa, H. Crystal structure of a heterotetrameric NMDA receptor ion channel. Science 344, 992–997 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kim, J.Y., Kim, C. & Lee, N.K. Real-time submillisecond single-molecule FRET dynamics of freely diffusing molecules with liposome tethering. Nat. Commun. 6, 6992 (2015).

    CAS  PubMed  Google Scholar 

  11. Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nat. Methods 5, 507–516 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Sasmal, D.K. & Lu, H.P. Single-molecule patch-clamp FRET microscopy studies of NMDA receptor ion channel dynamics in living cells: revealing the multiple conformational states associated with a channel at its electrical off state. J. Am. Chem. Soc. 136, 12998–13005 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Monyer, H. et al. Heteromeric NMDA receptors: molecular and functional distinction of subtypes. Science 256, 1217–1221 (1992).

    CAS  PubMed  Google Scholar 

  14. Traynelis, S.F. et al. Glutamate receptor ion channels: structure, regulation, and function. Pharmacol. Rev. 62, 405–496 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Kazi, R., Dai, J., Sweeney, C., Zhou, H.-X. & Wollmuth, L.P. Mechanical coupling maintains the fidelity of NMDA receptor-mediated currents. Nat. Neurosci. 17, 914–922 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Sobolevsky, A.I., Beck, C. & Wollmuth, L.P. Molecular rearrangements of the extracellular vestibule in NMDAR channels during gating. Neuron 33, 75–85 (2002).

    CAS  PubMed  Google Scholar 

  17. Borschel, W.F., Cummings, K.A., Tindell, L.K. & Popescu, G.K. Kinetic contributions to gating by interactions unique to N-methyl-D-aspartate (NMDA) receptors. J. Biol. Chem. 290, 26846–26855 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Amico-Ruvio, S.A., Murthy, S.E., Smith, T.P. & Popescu, G.K. Zinc effects on NMDA receptor gating kinetics. Biophys. J. 100, 1910–1918 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Sirrieh, R.E., MacLean, D.M. & Jayaraman, V. A conserved structural mechanism of NMDA receptor inhibition: A comparison of ifenprodil and zinc. J. Gen. Physiol. 146, 173–181 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Sirrieh, R.E., MacLean, D.M. & Jayaraman, V. Amino-terminal domain tetramer organization and structural effects of zinc binding in the N-methyl-D-aspartate (NMDA) receptor. J. Biol. Chem. 288, 22555–22564 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Sirrieh, R.E., MacLean, D.M. & Jayaraman, V. Subtype-dependent N-methyl-D-aspartate receptor amino-terminal domain conformations and modulation by spermine. J. Biol. Chem. 290, 12812–12820 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Rambhadran, A., Gonzalez, J. & Jayaraman, V. Conformational changes at the agonist binding domain of the N-methyl-D-aspartic acid receptor. J. Biol. Chem. 286, 16953–16957 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Cooper, D.R. et al. Conformational transitions in the glycine-bound GluN1 NMDA receptor LBD via single-molecule FRET. Biophys. J. 109, 66–75 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Dolino, D.M. et al. Structural dynamics of the glycine-binding domain of the N-methyl-D-aspartate receptor. J. Biol. Chem. 290, 797–804 (2015).

    CAS  PubMed  Google Scholar 

  25. Ramaswamy, S. et al. Role of conformational dynamics in α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor partial agonism. J. Biol. Chem. 287, 43557–43564 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Landes, C.F., Rambhadran, A., Taylor, J.N., Salatan, F. & Jayaraman, V. Structural landscape of isolated agonist-binding domains from single AMPA receptors. Nat. Chem. Biol. 7, 168–173 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Taylor, J.N., Makarov, D.E. & Landes, C.F. Denoising single-molecule FRET trajectories with wavelets and Bayesian inference. Biophys. J. 98, 164–173 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Taylor, J.N. & Landes, C.F. Improved resolution of complex single-molecule FRET systems via wavelet shrinkage. J. Phys. Chem. B 115, 1105–1114 (2011).

    CAS  PubMed  Google Scholar 

  29. Shuang, B. et al. Fast step transition and state identification (STaSI) for discrete single-molecule data analysis. J. Phys. Chem. Lett. 5, 3157–3161 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Chizhik, A.M. et al. Super-resolution optical fluctuation bio-imaging with dual-color carbon nanodots. Nano Lett. 16, 237–242 (2016).

    CAS  PubMed  Google Scholar 

  31. Hu, Z. et al. Excitonic energy migration in conjugated polymers: the critical role of interchain morphology. J. Am. Chem. Soc. 136, 16023–16031 (2014).

    CAS  PubMed  Google Scholar 

  32. Wedeking, T. et al. Single cell GFP-trap reveals stoichiometry and dynamics of cytosolic protein complexes. Nano Lett. 15, 3610–3615 (2015).

    CAS  PubMed  Google Scholar 

  33. Yang, J., Park, H. & Kaufman, L.J. Highly anisotropic conjugated polymer aggregates: preparation and quantification of physical and optical anisotropy. J. Phys. Chem. C 121, 13854–13862 (2017).

    CAS  Google Scholar 

  34. Zhang, W., Caldarola, M., Pradhan, B. & Orrit, M. Gold nanorod enhanced fluorescence enables single-molecule electrochemistry of methylene blue. Angew. Chem. Int. Ed. Engl. 56, 3566–3569 (2017).

    CAS  PubMed  Google Scholar 

  35. Alam, A. & Jiang, Y. High-resolution structure of the open NaK channel. Nat. Struct. Mol. Biol. 16, 30–34 (2009).

    CAS  PubMed  Google Scholar 

  36. Shi, N., Ye, S., Alam, A., Chen, L. & Jiang, Y. Atomic structure of a Na+- and K+-conducting channel. Nature 440, 570–574 (2006).

    CAS  PubMed  Google Scholar 

  37. Dolino, D.M., Rezaei Adariani, S., Shaikh, S.A., Jayaraman, V. & Sanabria, H. Conformational selection and submillisecond dynamics of the ligand-binding domain of the N-methyl-D-aspartate receptor. J. Biol. Chem. 291, 16175–16185 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Yao, Y., Belcher, J., Berger, A.J., Mayer, M.L. & Lau, A. Y. Conformational analysis of NMDA receptor GluN1, GluN2, and GluN3 ligand-binding domains reveals subtype-specific characteristics. Structure 21, 1788–1799 (2013).

    CAS  PubMed  Google Scholar 

  39. Peters, S., Koh, J. & Choi, D.W. Zinc selectively blocks the action of N-methyl-D-aspartate on cortical neurons. Science 236, 589–593 (1987).

    CAS  PubMed  Google Scholar 

  40. Low, C.M., Zheng, F., Lyuboslavsky, P. & Traynelis, S.F. Molecular determinants of coordinated proton and zinc inhibition of N-methyl-D-aspartate NR1/NR2A receptors. Proc. Natl. Acad. Sci. USA 97, 11062–11067 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Wong, E.H. et al. The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist. Proc. Natl. Acad. Sci. USA 83, 7104–7108 (1986).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Foster, A.C. & Wong, E.H. The novel anticonvulsant MK-801 binds to the activated state of the N-methyl-D-aspartate receptor in rat brain. Br. J. Pharmacol. 91, 403–409 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Lü, W., Du, J., Goehring, A. & Gouaux, E. Cryo-EM structures of the triheteromeric NMDA receptor and its allosteric modulation. Science 355, eaal3729 (2017).

    PubMed  PubMed Central  Google Scholar 

  44. 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. USA 85, 1307–1311 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Sobolevsky, A.I. Two-component blocking kinetics of open NMDA channels by organic cations. Biochim. Biophys. Acta 1416, 69–91 (1999).

    CAS  PubMed  Google Scholar 

  46. Sobolevsky, A. & Koshelev, S. Two blocking sites of amino-adamantane derivatives in open N-methyl-D-aspartate channels. Biophys. J. 74, 1305–1319 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Blanpied, T.A., Boeckman, F.A., Aizenman, E. & Johnson, J.W. Trapping channel block of NMDA-activated responses by amantadine and memantine. J. Neurophysiol. 77, 309–323 (1997).

    CAS  PubMed  Google Scholar 

  48. Mortensen, M. & Smart, T.G. Single-channel recording of ligand-gated ion channels. Nat. Protoc. 2, 2826–2841 (2007).

    CAS  PubMed  Google Scholar 

  49. Wang, J. & Wolynes, P. Instantons and the fluctuating path description of reactions in complex environments. J. Phys. Chem. 100, 1129–1136 (1996).

    CAS  Google Scholar 

  50. Pirchi, M. et al. Single-molecule fluorescence spectroscopy maps the folding landscape of a large protein. Nat. Commun. 2, 493 (2011).

    PubMed  Google Scholar 

  51. MacLean, D.M., Ramaswamy, S.S., Du, M., Howe, J.R. & Jayaraman, V. Stargazin promotes closure of the AMPA receptor ligand-binding domain. J. Gen. Physiol. 144, 503–512 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Shaikh, S.A. et al. Stargazin modulation of AMPA receptors. Cell Rep. 17, 328–335 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Jain, A. et al. Probing cellular protein complexes using single-molecule pull-down. Nature 473, 484–488 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Nick Taylor, J., Darugar, Q., Kourentzi, K., Willson, R.C. & Landes, C.F. Dynamics of an anti-VEGF DNA aptamer: a single-molecule study. Biochem. Biophys. Res. Commun. 373, 213–218 (2008).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Methods and additional data can be found in the supplementary materials. This project was supported by NIH grant R35GM122528 to V.J., K99NS094761to D.M.M., American Heart Association Fellowship 16POST30030007 to S.A.S., Schissler Foundation Fellowship to D.M.D., and Welch Foundation Grant C-1787 to C.F.L.

Author information

Author notes

  1. David M MacLean

    Present address: Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York, USA

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Center for Membrane Biology, University of Texas Health Science Center at Houston, Houston, Texas, USA

    Drew M Dolino, David M MacLean, Sana A Shaikh & Vasanthi Jayaraman

  2. Biochemistry and Molecular Biology Graduate Program, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, Texas, USA

    Drew M Dolino & Vasanthi Jayaraman

  3. Department of Chemistry, Rice University, Houston, Texas, USA

    Sudeshna Chatterjee, Charlotte Flatebo, Logan D C Bishop & Christy F Landes

  4. Department of Electrical and Computer Engineering, Rice University, Houston, Texas, USA

    Christy F Landes

Authors
  1. Drew M Dolino
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sudeshna Chatterjee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David M MacLean
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Charlotte Flatebo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Logan D C Bishop
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sana A Shaikh
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Christy F Landes
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Vasanthi Jayaraman
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.M.D. performed the mutations and prepared the labeled proteins, analyzed the smFRET data, and contributed to designing the research, interpreting the results, and writing the manuscript. S.C. performed the smFRET measurements, analyzed the smFRET data, and contributed to designing the research, interpreting the results, and writing the manuscript. D.M.M. and S.A.S. performed the electrophysiology. C.F. and L.D.C.B. analyzed the smFRET data. C.F.L., and V.J. analyzed the smFRET data and contributed to designing the research, interpreting the results, and writing the manuscript.

Corresponding authors

Correspondence to Christy F Landes or Vasanthi Jayaraman.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–7 (PDF 1105 kb)

Life Sciences Reporting Summary (PDF 90 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dolino, D., Chatterjee, S., MacLean, D. et al. The structure–energy landscape of NMDA receptor gating. Nat Chem Biol 13, 1232–1238 (2017). https://doi.org/10.1038/nchembio.2487

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2487

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing