Skip to main content
SpringerLink
Log in

Assaying the Energetics of NMDA Receptor Pore Opening

  • Protocol
Ionotropic Glutamate Receptor Technologies

Part of the book series: Neuromethods ((NM,volume 106))

Abstract

The NMDA receptor converts transient glutamate release into an electrical and biochemical signal that integrates neuronal activity and results in changes in synaptic connectivity and strength. A variety of approaches are used to address the linkage between neurotransmitter binding and ion channel opening, a key feature common to all ligand-gated ion channels. Φ-value or REFER analysis, an approach derived from chemical transition state theory, can characterize the energetics and dynamics of ion channel activation. Here, we describe an approach to apply Φ-value analysis to the unique features of NMDA receptor activity. The analysis is based on high-resolution single-channel recordings of single-channel patches and kinetic analysis. We further detail analytical approaches to performing burst analysis that is essential to accurately outline the multicomponent activation pathway leading to pore opening. Overall, this approach allows to quantitatively chart the allosteric cascade, at the level of single amino acids, as it propagates from the neurotransmitter-binding domain to the ion channel in NMDA receptors.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Notes

  1. 1.

    It is equally as possible that energy changes are a result of perturbations in the local environment and not isomerizations of the amino acid side-chain per se.

  2. 2.

    Too few or alike mutations may not adequately assay the conformational landscape of the position, thus resulting in underestimation of energetics and false negatives.

  3. 3.

    QuB uses an automated SKM algorithm [56] for data idealization while DCProgs uses a manual time course fitting approach. As such, QuB is better suited for high-throughput analysis such as required for energetic analyses.

  4. 4.

    False event rate and amplitude calculations are most accurately applied to data idealized using the half-amplitude threshold method.

  5. 5.

    Φ-values may also reflect a fractional structure of the side chain or that the side chain spends a certain fraction of the transition in a given end state. For example, a side-chain with a Φ-value of 0.9 is approximately 90 % isomerized or is isomerized 90 % of the time [35, 36, 48].

References

  1. Traynelis SF, Wollmuth LP, McBain CJ, Menniti FS et al (2010) Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev 62(3):405–496

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. daCosta CJ, Baenziger JE (2013) Gating of pentameric ligand-gated ion channels: structural insights and ambiguities. Structure 21(8):1271–1283

    Article  CAS  PubMed  Google Scholar 

  3. Lesca G, Rudolf G, Bruneau N, Lozovaya N et al (2013) GRIN2A mutations in acquired epileptic aphasia and related childhood focal epilepsies and encephalopathies with speech and language dysfunction. Nat Genet 45(9):1061–1066

    Article  CAS  PubMed  Google Scholar 

  4. Lemke JR, Lal D, Reinthaler EM, Steiner I et al (2013) Mutations in GRIN2A cause idiopathic focal epilepsy with rolandic spikes. Nat Genet 45(9):1067–1072

    Article  CAS  PubMed  Google Scholar 

  5. Hamdan FF, Gauthier J, Araki Y, Lin DT et al (2011) Excess of de novo deleterious mutations in genes associated with glutamatergic systems in nonsyndromic intellectual disability. Am J Hum Genet 88(3):306–316

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Steinlein OK, Mulley JC, Propping P, Wallace RH et al (1995) A missense mutation in the neuronal nicotinic acetylcholine receptor alpha 4 subunit is associated with autosomal dominant nocturnal frontal lobe epilepsy. Nat Genet 11(2):201–203

    Article  CAS  PubMed  Google Scholar 

  7. Wallace RH, Marini C, Petrou S, Harkin LA et al (2001) Mutant GABA(A) receptor gamma2-subunit in childhood absence epilepsy and febrile seizures. Nat Genet 28(1):49–52

    CAS  PubMed  Google Scholar 

  8. Popescu G, Auerbach A (2003) Modal gating of NMDA receptors and the shape of their synaptic response. Nat Neurosci 6(5):476–483

    CAS  PubMed  Google Scholar 

  9. Banke TG, Traynelis SF (2003) Activation of NR1/NR2B NMDA receptors. Nat Neurosci 6:144–152

    Article  CAS  PubMed  Google Scholar 

  10. Schorge S, Elenes S, Colquhoun D (2005) Maximum likelihood fitting of single channel NMDA activity with a mechanism composed of independent dimers of subunits. J Physiol 569(Pt 2):395–418

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Matouschek A, Kellis JT Jr, Serrano L, Fersht AR (1989) Mapping the transition state and pathway of protein folding by protein engineering. Nature 340(6229):122–126

    Article  CAS  PubMed  Google Scholar 

  12. Fersht AR, Sato S (2004) Phi-value analysis and the nature of protein-folding transition states. Proc Natl Acad Sci U S A 101(21):7976–7981

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Sunderman ER, Zagotta WN (1999) Sequence of events underlying the allosteric transition of rod cyclic nucleotide-gated channels. J Gen Physiol 113(5):621–640

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Grosman C, Zhou M, Auerbach A (2000) Mapping the conformational wave of acetylcholine receptor channel gating. Nature 403(6771):773–776

    Article  CAS  PubMed  Google Scholar 

  15. Zhou Y, Pearson JE, Auerbach A (2005) Phi-value analysis of a linear, sequential reaction mechanism: theory and application to ion channel gating. Biophys J 89(6):3680–3685

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Purohit P, Mitra A, Auerbach A (2007) A stepwise mechanism for acetylcholine receptor channel gating. Nature 446(7138):930–933

    Article  CAS  PubMed  Google Scholar 

  17. Kussius CL, Popescu GK (2009) Kinetic basis of partial agonism at NMDA receptors. Nat Neurosci 12(9):1114–1120

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Dingledine R, Borges K, Bowie D, Traynelis SF (1999) The glutamate receptor ion channels. Pharmacol Rev 51:7–61

    CAS  PubMed  Google Scholar 

  19. Karakas E, Furukawa H (2014) Crystal structure of a heterotetrameric NMDA receptor ion channel. Science 344(6187):992–997

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Lee CH, Lu W, Michel JC, Goehring A et al (2014) NMDA receptor structures reveal subunit arrangement and pore architecture. Nature 511(7508):191–197

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Armstrong N, Gouaux E (2000) Mechanisms for activation and antagonism of an AMPA-sensitive glutamate receptor: crystal structures of the GluR2 ligand binding core. Neuron 28:165–181

    Article  CAS  PubMed  Google Scholar 

  22. Sun Y, Olson R, Horning M, Armstrong N et al (2002) Mechanism of glutamate receptor desensitization. Nature 417:245–253

    Article  CAS  PubMed  Google Scholar 

  23. Mayer ML, Olson R, Gouaux E (2001) Mechanisms for ligand binding to GluR0 ion channels: crystal structures of the glutamate and serine complexes and a closed apo state. J Mol Biol 311:815–836

    Article  CAS  PubMed  Google Scholar 

  24. Furukawa H, Singh SK, Mancusso R, Gouaux E (2005) Subunit arrangement and function in NMDA receptors. Nature 438(7065):185–192

    Article  CAS  PubMed  Google Scholar 

  25. Yao Y, Belcher J, Berger AJ, Mayer ML et al (2013) Conformational analysis of NMDA receptor GluN1, GluN2, and GluN3 ligand-binding domains reveals subtype-specific characteristics. Structure 21(10):1788–1799

    Article  CAS  PubMed  Google Scholar 

  26. Lau AY, Roux B (2011) The hidden energetics of ligand binding and activation in a glutamate receptor. Nat Struct Mol Biol 18(3):283–287

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Motlagh HN, Wrabl JO, Li J, Hilser VJ (2014) The ensemble nature of allostery. Nature 508(7496):331–339

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Talukder I, Borker P, Wollmuth LP (2010) Specific sites within the ligand-binding domain and ion channel linkers modulate NMDA receptor gating. J Neurosci 30(35):11792–11804

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Sobolevsky AI, Prodromou ML, Yelshansky MV, Wollmuth LP (2007) Subunit-specific contribution of pore-forming domains to NMDA receptor channel structure and gating. J Gen Physiol 129(6):509–525

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Chang HR, Kuo CC (2008) The activation gate and gating mechanism of the NMDA receptor. J Neurosci 28(7):1546–1556

    Article  CAS  PubMed  Google Scholar 

  31. Ramaswamy S, Cooper D, Poddar N, MacLean DM et al (2012) Role of conformational dynamics in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor partial agonism. J Biol Chem 287(52):43557–43564

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Landes CF, Rambhadran A, Taylor JN, Salatan F et al (2011) Structural landscape of isolated agonist-binding domains from single AMPA receptors. Nat Chem Biol 7(3):168–173

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. Lau AY, Salazar H, Blachowicz L, Ghisi V et al (2013) A conformational intermediate in glutamate receptor activation. Neuron 79(3):492–503

    Article  CAS  PubMed  Google Scholar 

  34. Schauder DM, Kuybeda O, Zhang J, Klymko K et al (2013) Glutamate receptor desensitization is mediated by changes in quaternary structure of the ligand binding domain. Proc Natl Acad Sci U S A 110(15):5921–5926

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Fersht AR (2004) Relationship of Leffler (Bronsted) alpha values and protein folding Phi values to position of transition-state structures on reaction coordinates. Proc Natl Acad Sci U S A 101(40):14338–14342

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Auerbach A (2007) How to turn the reaction coordinate into time. J Gen Physiol 130(6):543–546

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  37. Ozkan SB, Bahar I, Dill KA (2001) Transition states and the meaning of Phi-values in protein folding kinetics. Nat Struct Biol 8(9):765–769

    Article  CAS  PubMed  Google Scholar 

  38. Fersht AR, Daggett V (2002) Protein folding and unfolding at atomic resolution. Cell 108(4):573–582

    Article  CAS  PubMed  Google Scholar 

  39. Raleigh DP, Plaxco KW (2005) The protein folding transition state: what are Phi-values really telling us? Protein Pept Lett 12(2):117–122

    Article  CAS  PubMed  Google Scholar 

  40. Merlo C, Dill KA, Weikl TR (2005) Phi values in protein-folding kinetics have energetic and structural components. Proc Natl Acad Sci U S A 102(29):10171–10175

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  41. Purohit P, Gupta S, Jadey S, Auerbach A (2013) Functional anatomy of an allosteric protein. Nat Commun 4:2984

    Article  PubMed Central  PubMed  Google Scholar 

  42. Wang DT, Hill AP, Mann SA, Tan PS et al (2011) Mapping the sequence of conformational changes underlying selectivity filter gating in the K(v)11.1 potassium channel. Nat Struct Mol Biol 18(1):35–41

    Article  PubMed  Google Scholar 

  43. Purohit P, Auerbach A (2009) Unliganded gating of acetylcholine receptor channels. Proc Natl Acad Sci U S A 106(1):115–120

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  44. Grosman C, Auerbach A (2000) Kinetic, mechanistic, and structural aspects of unliganded gating of acetylcholine receptor channels: a single-channel study of second transmembrane segment 12′ mutants. J Gen Physiol 115(5):621–635

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  45. Jackson MB (1986) Kinetics of unliganded acetylcholine receptor channel gating. Biophys J 49(3):663–672

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  46. Monod J, Wyman J, Changeux JP (1965) On the nature of allosteric transitions: a plausible model. J Mol Biol 12:88–118

    Article  CAS  PubMed  Google Scholar 

  47. Auerbach A (2012) Thinking in cycles: MWC is a good model for acetylcholine receptor-channels. J Physiol 590(Pt 1):93–98

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  48. Auerbach A (2005) Gating of acetylcholine receptor channels: Brownian motion across a broad transition state. Proc Natl Acad Sci U S A 102(5):1408–1412

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  49. Gibb AJ, Colquhoun D (1992) Activation of N-methyl-D-aspartate receptors by L-glutamate in cells dissociated from adult rat hippocampus. J Physiol 456(143):143–179

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  50. Stern P, Cik M, Colquhoun D, Stephenson FA (1994) Single channel properties of cloned NMDA receptors in a human cell line: comparison with results from Xenopus oocytes. J Physiol 476(3):391–397

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  51. Auerbach A, Zhou Y (2005) Gating reaction mechanisms for NMDA receptor channels. J Neurosci 25:7914–7923

    Article  PubMed  Google Scholar 

  52. Auerbach A (2013) The energy and work of a ligand-gated ion channel. J Mol Biol 425(9):1461–1475

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  53. Maki BA, Cummings KA, Paganelli MA, Murthy SE et al (2014) One-channel cell-attached patch-clamp recording. J Vis Exp 88. doi:10.3791/51629

  54. Kazi R, Dai J, Sweeney C, Zhou HX et al (2014) Mechanical coupling maintains the fidelity of NMDA receptor-mediated currents. Nat Neurosci 17(7):914–922

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  55. Nicolai C, Sachs F (2013) Solving ion channel kinetics with the QuB software. Biophys Rev Lett 08(03):1–21

    Google Scholar 

  56. Qin F (2004) Restoration of single-channel currents using the segmental k-means method based on hidden Markov modeling. Biophys J 86(3):1488–1501

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  57. Howe JR, Cull-Candy SG, Colquhoun D (1991) Currents through single glutamate receptor channels in outside-out patches from rat cerebellar granule cells. J Physiol 432:143–202

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  58. Colquhoun D, Sigworth FJ (1995) Fitting and statistical analysis of single-channel records. In: Sakmann B, Neher E (eds) Single-channel recording. Plenum, New York, pp 397–482

    Chapter  Google Scholar 

  59. Qin F, Auerbach A, Sachs F (2000) Hidden Markov modeling for single channel kinetics with filtering and correlated noise. Biophys J 79(4):1928–1944

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  60. Popescu G, Robert A, Howe JR, Auerbach A (2004) Reaction mechanism determines NMDA receptor response to repetitive stimulation. Nature 430(7001):790–793

    Article  CAS  PubMed  Google Scholar 

  61. Zhang W, Howe JR, Popescu GK (2008) Distinct gating modes determine the biphasic relaxation of NMDA receptor currents. Nat Neurosci 11(12):1373–1375

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  62. Talukder I, Wollmuth LP (2011) Local constraints in either the GluN1 or GluN2 subunit equally impair NMDA receptor pore opening. J Gen Physiol 138(2):179–194

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  63. Kazi R, Gan Q, Talukder I, Markowitz M et al (2013) Asynchronous movements prior to pore opening in NMDA receptors. J Neurosci 33(29):12052–12066

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  64. Talukder I, Kazi R, Wollmuth LP (2011) GluN1-specific redox effects on the kinetic mechanism of NMDA receptor activation. Biophys J 101(10):2389–2398

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  65. Magleby KL, Pallotta BS (1983) Burst kinetics of single calcium-activated potassium channels in cultured rat muscle. J Physiol 344:605–623

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  66. Qin F, Auerbach A, Sachs F (2000) A direct optimization approach to hidden Markov modeling for single channel kinetics. Biophys J 79(4):1915–1927

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  67. Purohit Y, Grosman C (2006) Estimating binding affinities of the nicotinic receptor for low-efficacy ligands using mixtures of agonists and two-dimensional concentration-response relationships. J Gen Physiol 127(6):719–735

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  68. Qin F, Auerbach A, Sachs F (1996) Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events. Biophys J 70(1):264–280

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  69. Qin F, Auerbach A, Sachs F (1997) Maximum likelihood estimation of aggregated Markov processes. Proc Biol Sci 264(1380):375–383

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  70. Colquhoun D, Hawkes AG (1990) Stochastic properties of ion channel openings and bursts in a membrane patch that contains two channels: evidence concerning the number of channels present when a record containing only single openings is observed. Proc R Soc Lond Ser B, containing papers of a biological character. Royal Society. 240(1299): 453–477

    Google Scholar 

  71. Dravid SM, Prakash A, Traynelis SF (2008) Activation of recombinant NR1/NR2C NMDA receptors. J Physiol 586(Pt 18):4425–4439

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Drs. Prasad Purohit and Anthony Auerbach and Quan Gan for helpful discussions and/or comments on the chapter.

This work was supported by an NIH RO1 grants from NINDS (NS088479, L.P.W.), a Hartman Foundation for Parkinson’s research (L.P.W.), a URECA-Biology Alumni (UBAR) Award (M.D.), and an NIH NRSA from NINDS (NS0777541) (R.K.).

Author information

Authors and Affiliations

  1. Medical Scientist Training Program (MSTP), Stony Brook University, Stony Brook, New York, NY, USA

    Rashek Kazi

  2. Graduate Program in Neuroscience, Stony Brook University, Stony Brook, New York, NY, USA

    Rashek Kazi

  3. Center for Nervous System Disorders, Stony Brook University, Stony Brook, New York, NY, USA

    Rashek Kazi, Melissa Daniel & Lonnie P. Wollmuth

  4. Department of Neurobiology and Behavior, Stony Brook University, Stony Brook, New York, NY, USA

    Rashek Kazi, Melissa Daniel & Lonnie P. Wollmuth

Authors
  1. Rashek Kazi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Melissa Daniel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lonnie P. Wollmuth
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lonnie P. Wollmuth .

Editor information

Editors and Affiliations

  1. Department of Biochemistry, University at Buffalo, Buffalo, New York, USA

    Gabriela K. Popescu

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Kazi, R., Daniel, M., Wollmuth, L.P. (2016). Assaying the Energetics of NMDA Receptor Pore Opening. In: Popescu, G. (eds) Ionotropic Glutamate Receptor Technologies. Neuromethods, vol 106. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2812-5_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2812-5_11

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2811-8

  • Online ISBN: 978-1-4939-2812-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation