Abstract
Like all neurotransmitter-gated channels, in response to agonist binding, ionotropic glutamate receptors produce electrical signals whose amplitudes and durations reflect intramolecular transitions between non-conducting (closed) and conducting (open) receptor conformations. Thus, delineating the reaction mechanism of synaptic channels represents an important step in understanding how information is transferred and processed in the nervous system. The recorded single-channel signal captures in real-time multiple series of discrete current amplitudes, whose complex duration distributions contain valuable information about the underlying kinetic mechanism but in most cases are difficult to decipher. For NMDA receptors, we identified conditions in which the receptor populates only two conductance levels, corresponding to closed and open channels, and we developed procedures that can organize the entire succession of closed and open durations into a comprehensive, reproducible, and testable reaction mechanism. In this chapter, we describe how to select, process, and idealize current traces recorded from patches containing one NMDA receptor, and how to build increasingly more accurate kinetic models that include transitions from the sub-millisecond to the hundreds of minutes time scales. The resulting schemes can be tested by comparing model simulations and experimental recordings elicited with similar stimulation patterns. The principles and methodology outlined here can be adapted and extended to other ion channels to gather deeper insight into the order and rates of intramolecular movements that produce stimulus-elicited electrical signals in the central nervous system.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bean RC, Shepherd WC, Chan H, Eichner J (1969) Discrete conductance fluctuations in lipid bilayer protein membranes. J Gen Physiol 53(6):741–757
Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391(2):85–100
Neher E (1981) Unit conductance studies in biological membranes. In: Baker PF (ed)Techniques in Cellular Physiology. pp. 1–32, Elsevier, Amsterdam
Neher E, Sakmann B (1976) Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature 260(5554):799–802
Sigworth FJ, Neher E (1980) Single Na+ channel currents observed in cultured rat muscle cells. Nature 287(5781):447–449
Kapanidis AN, Strick T (2009) Biology, one molecule at a time. Trends Biochem Sci 34(5):234–243
Nie S, Zare RN (1997) Optical detection of single molecules. Annu Rev Biophys Biomol Struct 26:567–596
Neher E, Sakmann B, Steinbach JH (1978) The extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes. Pflugers Arch 375(2):219–228
Neher E, Steinbach JH (1978) Local anaesthetics transiently block currents through single acetylcholine-receptor channels. J Physiol 277:153–176
Colquhoun D, Hawkes AG (1981) On the stochastic properties of single ion channels. Proc R Soc Lond B Biol Sci 211(1183):205–235
Colquhoun D, Hawkes AG (1982) On the stochastic properties of bursts of single ion channel openings and of clusters of bursts. Philos Trans R Soc Lond B Biol Sci 300(1098):1–59
Maki BA, Cummings KA, Paganelli MA, Murthy SE, Popescu GK (2014) One-channel cell-attached patch-clamp recording. J Vis Exp 9(88)
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
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
Popescu G (2005) Mechanism-based targeting of NMDA receptor functions. Cell Mol Life Sci 62(18):2100–2111
Popescu G (2005) Principles of N-methyl-D-aspartate receptor allosteric modulation. Mol Pharmacol 68(4):1148–1155
Amico-Ruvio S, Popescu G (2010) Stationary gating of GluN1/GluN2B receptors in intact membrane patches. Biophys J 98(7):1160–1169
Borschel WF, Myers JM, Kasperek EM, Smith TP, Graziane NM, Nowak LM, Popescu GK (2012) Gating reaction mechanism of neuronal NMDA receptors. J Neurophysiol 108(11):3105–3115
Kussius CL, Popescu GK (2009) Kinetic basis of partial agonism at NMDA receptors. Nat Neurosci 12(9):1114–1120
Popescu G, Robert A, Howe JR, Auerbach A (2004) Reaction mechanism determines NMDA receptor response to repetitive stimulation. Nature 430(7001):790–793
Zhang W, Howe JR, Popescu GK (2008) Distinct gating modes determine the biphasic relaxation of NMDA receptor currents. Nat Neurosci 11(12):1373–1375
Colquhoun D, Hatton CJ, Hawkes AG (2003) The quality of maximum likelihood estimates of ion channel rate constants. J Physiol 547(Pt 3):699–728
Colquhoun D, Hawkes AG, Srodzinski K (1996) Joint distributions of apparent open and shut times of single-ion channels and maximum likelihood fitting of mechanisms. Philos Trans Math Phys Eng Sci 354(1718):2555–2590
Hawkes AG, Jalali A, Colquhoun D (1990) The distributions of the apparent open times and shut times in a single channel record when brief events cannot be detected. Philos Trans Phys Sci Eng 332(1627):511–538
Hawkes AG, Jalali A, Colquhoun D (1992) Asymptotic distributions of apparent open times and shut times in a single channel record allowing for the omission of brief events. Philos Trans R Soc Lond B Biol Sci 337(1282):383–404
Qin F (2014) Principles of single-channel kinetic analysis. In: Martina M, Taverna S (eds) Patch-clamp methods and protocols, vol 1183. Springer, New York, pp 371–399
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
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
Nicolai C, Sachs F (2013) Solving ion channel kinetics with the QuB software. Biophys Rev Lett 08(03n04):191–211
Ascher P, Bregestovski P, Nowak L (1988) NMDA-activated channels of mouse central neurones in magnesium-free solutions. J Physiol 399:207–226
Nowak L, Bregestovski P, Ascher P, Herbet A, Prochiantz A (1984) Magnesium gates glutamate-activated channels in mouse central neurones. Nature 307(5950):462–465
Traynelis SF, Cull-Candy SG (1990) Proton inhibition of N-methyl-D-aspartate receptors in cerebellar neurons. Nature 345(6273):347–350
Anson LC, Chen PE, Wyllie DJA, Colquhoun D, Schoepfer R (1998) Identification of amino acid residues of the NR2A subunit that control glutamate potency in recombinant NR1/NR2A NMDA receptors. J Neurosci 18(2):581–589
Howe JR, Colquhoun D, Cull-Candy SG (1988) On the kinetics of large-conductance glutamate-receptor ion channels in rat cerebellar granule neurons. Proc R Soc Lond B Biol Sci 233(1273):407–422
Wyllie DJ, Behe P, Colquhoun D (1998) Single-channel activations and concentration jumps: comparison of recombinant NR1a/NR2A and NR1a/NR2D NMDA receptors. J Physiol 510(Pt 1):1–18
Anson LC, Schoepfer R, Colquhoun D, Wyllie DJ (2000) Single-channel analysis of an NMDA receptor possessing a mutation in the region of the glutamate binding site. J Physiol 527(Pt 2):225–237
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 B Biol Sci 240(1299):453–477
Erreger K, Dravid SM, Banke TG, Wyllie DJ, Traynelis SF (2005) Subunit-specific gating controls rat NR1/NR2A and NR1/NR2B NMDA channel kinetics and synaptic signalling profiles. J Physiol 563(Pt 2):345–358
Popescu G, Auerbach A (2003) Modal gating of NMDA receptors and the shape of their synaptic response. Nat Neurosci 6(5):476–483
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
Kussius CL, Kaur N, Popescu GK (2009) Pregnanolone sulfate promotes desensitization of activated NMDA receptors. J Neurosci 29(21):6819–6827
Popescu G, Auerbach A (2004) The NMDA receptor gating machine: lessons from single channels. Neuroscientist 10(3):192–198
Suchyna TM, Markin VS, Sachs F (2009) Biophysics and structure of the patch and the gigaseal. Biophys J 97(3):738–747
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
Qin F, Auerbach A, Sachs F (1997) Maximum likelihood estimation of aggregated Markov processes. Proc Biol Sci 264(1380):375–383
Qin F, Li L (2004) Model-based fitting of single-channel dwell-time distributions. Biophys J 87(3):1657–1671
Horn R (1987) Statistical methods for model discrimination. Applications to gating kinetics and permeation of the acetylcholine receptor channel. Biophys J 51(2):255–263
Popescu GK (2012) Modes of glutamate receptor gating. J Physiol 590(1):73–91
Borschel WF, Murthy SE, Kasperek EM, Popescu GK (2011) NMDA receptor activation requires remodelling of intersubunit contacts within ligand-binding heterodimers. Nat Commun 2:498
Auerbach A, Zhou Y (2005) Gating reaction mechanisms for NMDA receptor channels. J Neurosci 25(35):7914–7923
Akaike H (1974) A new look at the statistical model identification. IEEE Trans Autom Control 19(6):716–723
Acknowledgements
This work was supported by grants from the National Institutes of Health: RO1NS052669 (to G.K.P.) and F31NS086765 (to K.A.C.); and from the American Heart Association: EIA9100012 (to G.K.P.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Cummings, K.A., Iacobucci, G.J., Popescu, G.K. (2016). Extracting Rate Constants for NMDA Receptor Gating from One-Channel Current Recordings. 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_18
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2812-5_18
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