Skip to main content
SpringerLink
Log in

Analysis of Whole-Cell NMDA Receptor Currents

  • Protocol
Book cover Ionotropic Glutamate Receptor Technologies

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

Abstract

During receptor channel activation, the receptor goes through a ligand binding step followed by conformation steps leading to the open channel conformation. The probability of finding the receptor in a particular conformation is given by the apparent rate constants. The apparent rate constants describe the rate of transitions between conformational states and vary among different types of iGluR. To determine these parameters reflecting receptor channel function, several approaches have been developed. In this chapter we describe methods for the measurement of basic kinetic parameters of the NMDA receptor determined from non-stationary analysis of whole-cell currents. Particularly, the apparent rate constants of agonist binding/unbinding, desensitization/resensitization, and the probability of channel opening are determined. Besides the kinetic parameters of the receptor, the number of ion channels in the cell membrane and single-channel conductance are estimated from whole-cell recordings.

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

References

  1. 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(1):165–181

    Article  CAS  PubMed  Google Scholar 

  2. Mayer ML (2005) Crystal structures of the GluR5 and GluR6 ligand binding cores: molecular mechanisms underlying kainate receptor selectivity. Neuron 45(4):539–552

    Article  CAS  PubMed  Google Scholar 

  3. 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 

  4. Meyerson JR et al (2014) Structural mechanism of glutamate receptor activation and desensitization. Nature 514:328–334

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Lee C-H et al (2014) NMDA receptor structures reveal subunit arrangement and pore architecture. Nature 511(7508):191–197

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Mayer ML (2006) Glutamate receptors at atomic resolution. Nature 440(7083):456–462

    Article  CAS  PubMed  Google Scholar 

  7. Mayer ML, Armstrong N (2004) Structure and function of glutamate receptor ion channels. Annu Rev Physiol 66:161–181

    Article  CAS  PubMed  Google Scholar 

  8. Furukawa H et al (2005) Subunit arrangement and function in NMDA receptors. Nature 438(7065):185–192

    Article  CAS  PubMed  Google Scholar 

  9. Hamill OP et al (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391(2):85–100

    Article  CAS  PubMed  Google Scholar 

  10. Sakmann B, Neher E (1984) Patch clamp techniques for studying ionic channels in excitable membranes. Annu Rev Physiol 46:455–472

    Article  CAS  PubMed  Google Scholar 

  11. Vyklicky V et al (2013) Structure, function, and pharmacology of NMDA receptor channels. Physiol Res 63:S191–S203

    Google Scholar 

  12. Molecular Devices Corporation (2006) The axon CNS guide to electrophysiology & biophysics laboratory techniques. Molecular Devices Corporation, Union City

    Google Scholar 

  13. Molnar P, Hickman JJ (2007) Patch-clamp methods and protocols. Humana, New York

    Book  Google Scholar 

  14. Sachs F (1999) Practical limits on the maximal speed of solution exchange for patch clamp experiments. Biophys J 77(2):682–690

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Auzmendi J et al (2012) Achieving maximal speed of solution exchange for patch clamp experiments. PloS one 7(8):e42275

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Lester RA, Jahr CE (1992) NMDA channel behavior depends on agonist affinity. J Neurosci 12(2):635–643

    CAS  PubMed  Google Scholar 

  17. Popescu G et al (2004) Reaction mechanism determines NMDA receptor response to repetitive stimulation. Nature 430(7001):790–793

    Article  CAS  PubMed  Google Scholar 

  18. Auerbach A, Zhou Y (2005) Gating reaction mechanisms for NMDA receptor channels. J Neurosci 25(35):7914–7923

    Article  PubMed  Google Scholar 

  19. Kussius CL, Kaur N, Popescu GK (2009) Pregnanolone sulfate promotes desensitization of activated NMDA receptors. J Neurosci 29(21):6819–6827

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Huettner JE, Bean BP (1988) 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 85(4):1307–1311

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Jahr CE (1992) High probability opening of NMDA receptor channels by L-glutamate. Science 255(5043):470–472

    Article  CAS  PubMed  Google Scholar 

  22. Rosenmund C, Feltz A, Westbrook GL (1995) Synaptic NMDA receptor channels have a low open probability. J Neurosci 15(4):2788–2795

    CAS  PubMed  Google Scholar 

  23. Gielen M et al (2009) Mechanism of differential control of NMDA receptor activity by NR2 subunits. Nature 459(7247):703–707

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Nowak L et al (1984) Magnesium gates glutamate-activated channels in mouse central neurones. Nature 307(5950):462–465

    Article  CAS  PubMed  Google Scholar 

  25. Usowicz MM, Gallo V, Cull Candy SG (1989) Multiple conductance channels in type-2 cerebellar astrocytes activated by excitatory amino acids. Nature 339(6223):380–383

    Article  CAS  PubMed  Google Scholar 

  26. Stern P et al (1992) Single-channel conductances of NMDA receptors expressed from cloned cDNAs: comparison with native receptors. Proc R Soc Lond B Biol Sci 250(1329):271–277

    Article  CAS  Google Scholar 

  27. Stern P et al (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 

  28. Wyllie DJ et al (1996) Single-channel currents from recombinant NMDA NR1a/NR2D receptors expressed in Xenopus oocytes. Proc R Soc Lond B Biol Sci 263(1373):1079–1086

    Article  CAS  Google Scholar 

  29. Clark BA, Farrant M, Cull-Candy SG (1997) A direct comparison of the single-channel properties of synaptic and extrasynaptic NMDA receptors. J Neurosci 17(1):107–116

    CAS  PubMed  Google Scholar 

  30. Robinson HP, Sahara Y, Kawai N (1991) Nonstationary fluctuation analysis and direct resolution of single channel currents at postsynaptic sites. Biophys J 59(2):295–304

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  31. Sigworth FJ (1980) The variance of sodium current fluctuations at the node of Ranvier. J Physiol 307:97–129

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Abdrachmanova G et al (2000) Molecular and functional properties of synaptically activated NMDA receptors in neonatal motoneurons in rat spinal cord slices. Eur J Neurosci 12(3):955–963

    Article  CAS  PubMed  Google Scholar 

  33. Silver RA, Traynelis SF, Cull Candy SG (1992) Rapid-time-course miniature and evoked excitatory currents at cerebellar synapses in situ. Nature 355(6356):163–166

    Article  CAS  PubMed  Google Scholar 

  34. Vyklicky L Jr (1993) Calcium-mediated modulation of N-methyl-D-aspartate (NMDA) responses in cultured rat hippocampal neurones. J Physiol 470:575–600

    Article  PubMed Central  CAS  PubMed  Google Scholar 

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

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Cais O et al (2008) Temperature dependence of NR1/NR2B NMDA receptor channels. Neuroscience 151(2):428–438

    Article  CAS  PubMed  Google Scholar 

  37. Sather W et al (1992) Activation and desensitization of N-methyl-D-aspartate receptors in nucleated outside-out patches from mouse neurones. J Physiol 450:643–672

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Work on this chapter was supported by the GACR (P303/12/1464, P303/11/P391), RP (RVO: 67985823), TACR (TE01020028), BIOCEV (CZ.1.05/1.1.00/02.0109), and GAUK 800313/2012/2.lF. We thank J. Krusek for helpful discussions and commentary.

Author information

Authors and Affiliations

  1. Institute of Physiology, AS CR, Videnska 1083, Prague 4, 142 20, Czech Republic

    Vojtech Vyklicky, Miloslav Korinek, Ales Balik, Tereza Smejkalova, Barbora Krausova & Ladislav Vyklicky

Authors
  1. Vojtech Vyklicky
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Miloslav Korinek
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ales Balik
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tereza Smejkalova
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Barbora Krausova
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ladislav Vyklicky
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ladislav Vyklicky .

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

Vyklicky, V., Korinek, M., Balik, A., Smejkalova, T., Krausova, B., Vyklicky, L. (2016). Analysis of Whole-Cell NMDA Receptor Currents. 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_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2812-5_14

  • 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