Abstract
Electrophysiological recording of single-channel currents is the most direct method available for obtaining detailed and precise information about the kinetic behavior of ion channels. A wide variety of cell types can be used for single-channel recording, but to obtain the highest resolution of the briefest channel opening and closing events, low-noise recordings, coupled with a minimal filtering frequency, are required. Here, we present a protocol designed to help those with some electrophysiological expertise who wish to explore the properties of native and recombinant single ligand-gated ion channels. We have focused on the practical aspects of recording single GABA channels from cell-attached and outside-out patches and also introduced some of the preliminary considerations that are necessary for the analysis of single-channel data, including an introduction to single-channel analysis software.
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References
Ehrenstein, G., Lecar, H. & Nossal, R. The nature of the negative resistance in bimolecular lipid membranes containing excitability-inducing material. J. Gen. Physiol. 55, 119–133 (1970).
Hladky, S.B. & Haydon, D.A. Discreteness of conductance change in bimolecular lipid membranes in the presence of certain antibiotics. Nature 225, 451–453 (1970).
Katz, B. & Miledi, R. The statistical nature of the acetycholine potential and its molecular components. J. Physiol. 224, 665–699 (1972).
Neher, E. & Sakmann, B. Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature 260, 799–802 (1976).
Neher, E., Sakmann, B. & Steinbach, J.H. The extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes. Pflugers Arch. 375, 219–228 (1978).
Patlak, J.B., Gration, K.A. & Usherwood, P.N. Single glutamate-activated channels in locust muscle. Nature 278, 643–645 (1979).
Sigworth, F.J. & Neher, E. Single Na+ channel currents observed in cultured rat muscle cells. Nature 287, 447–449 (1980).
Hamill, O.P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F.J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391, 85–100 (1981).
Verheugen, J.A., Fricker, D. & Miles, R. Noninvasive measurements of the membrane potential and GABAergic action in hippocampal interneurons. J. Neurosci. 19, 2546–2555 (1999).
Sigworth, F.J. The variance of sodium current fluctuations at the node of Ranvier. J. Physiol. 307, 97–129 (1980).
Hocherman, S.D. & Bezanilla, F. A patch-clamp study of delayed rectifier currents in skeletal muscle of control and mdx mice. J. Physiol. 493, 113–128 (1996).
Traynelis, S.F., Silver, R.A. & Cull-Candy, S.G. Estimated conductance of glutamate receptor channels activated during EPSCs at the cerebellar mossy fiber-granule cell synapse. Neuron 11, 279–289 (1993).
De Koninck, Y. & Mody, I. Noise analysis of miniature IPSCs in adult rat brain slices: properties and modulation of synaptic GABAA receptor channels. J. Neurophysiol. 71, 1318–1335 (1994).
Benke, T.A. et al. Mathematical modelling of non-stationary fluctuation analysis for studying channel properties of synaptic AMPA receptors. J. Physiol. 537, 407–420 (2001).
Hartveit, E. & Veruki, M.L. Studying properties of neurotransmitter receptors by non-stationary noise analysis of spontaneous postsynaptic currents and agonist-evoked responses in outside-out patches. Nat. Protoc. 2, 434–448 (2007).
Thomas, P. & Smart, T.G. HEK293 cell line: a vehicle for the expression of recombinant proteins. J. Pharmacol. Toxicol. Methods 51, 187–200 (2005).
Thomas, P. & Smart, T.G. Current Protocols in Pharmacology (eds. Enna, S.J. et al.), 11.4.1–11.4.34 (John Wiley & Sons, New Jersey, 2002).
Groot-Kormelink, P.J., Beato, M., Finotti, C., Harvey, R.J. & Sivilotti, L.G. Achieving optimal expression for single channel recording: a plasmid ratio approach to the expression of alpha 1 glycine receptors in HEK293 cells. J. Neurosci. Methods 113, 207–214 (2002).
Mortensen, M. et al. Activation of single heteromeric GABA(A) receptor ion channels by full and partial agonists. J. Physiol. 557, 389–413 (2004).
Colquhoun, D. & Sigworth, F.J. Single-Channel Recording (eds. Sakmann, B. & Neher, E.) 483–587 (Plenum Press, New York, 1995).
Sakmann, B. & Neher, E. Single-Channel Recording (eds. Sakmann, B. & Neher, E.) (Plenum Press, New York, 1995).
Dempster, J. Computer Analysis of Electrophysiological Signals (Academic Press, London, 1993).
Sigworth, F.J. & Sine, S.M. Data transformations for improved display and fitting of single-channel dwell time histograms. Biophys. J. 52, 1047–1054 (1987).
Colquhoun, D. & Sakmann, B. Fast events in single-channel currents activated by acetylcholine and its analogues at the frog muscle end-plate. J. Physiol. 369, 501–557 (1985).
Hatton, C.J., Shelley, C., Brydson, M., Beeson, D. & Colquhoun, D. Properties of the human muscle nicotinic receptor, and of the slow-channel myasthenic syndrome mutant εL221F, inferred from maximum likelihood fits. J. Physiol. 547, 729–760 (2003).
Burzomato, V., Beato, M., Groot-Kormelink, P.J., Colquhoun, D. & Sivilotti, L.G. Single-channel behavior of heteromeric α1β glycine receptors: an attempt to detect a conformational change before the channel opens. J. Neurosci. 24, 10924–10940 (2004).
Popescu, G. & Auerbach, A. Modal gating of NMDA receptors and the shape of their synaptic response. Nat. Neurosci. 6, 476–483 (2003).
Lema, G.M. & Auerbach, A. Modes and models of GABAA receptor gating. J. Physiol. 572, 183–200 (2006).
Celentano, J.J. & Wong, R.K. Multiphasic desensitization of the GABAA receptor in outside-out patches. Biophys. J. 66, 1039–1050 (1994).
MacDonald, R.L., Rogers, C.J. & Twyman, R.E. Kinetic properties of the GABAA receptor main conductance state of mouse spinal cord neurones in culture. J. Physiol. (Lond.) 410, 479–499 (1989).
Jones, M.V. & Westbrook, G.L. Desensitized states prolong GABAA channel responses to brief agonist pulses. Neuron 15, 181–191 (1995).
Haas, K.F. & MacDonald, R.L. GABAA receptor subunit γ2 and δ subtypes confer unique kinetic properties on recombinant GABAA receptor currents in mouse fibroblasts. J. Physiol. 514, 27–45 (1999).
Steinbach, J.H. & Akk, G. Modulation of GABAA receptor channel gating by pentobarbital. J. Physiol. 537, 715–733 (2001).
Acknowledgements
We thank Marco Beato for comments on the manuscript and Guy Moss for discussion. This work was supported by MRC and the Alfred Benzon's Foundation.
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Mortensen, M., Smart, T. Single-channel recording of ligand-gated ion channels. Nat Protoc 2, 2826–2841 (2007). https://doi.org/10.1038/nprot.2007.403
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DOI: https://doi.org/10.1038/nprot.2007.403
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