Cell-attached patch-clamp recording
Materials Required
Principle
Cell-attached patch-clamp recording measures ionic current through one or more ion channels in a small membrane patch that remains attached to an intact cell; the readout is a time-resolved current trace generated when channels in the sealed patch open and close under controlled pipette voltage or stimulus conditions[1][2][3]. Classic applications include single acetylcholine receptor currents in frog skeletal muscle, single sodium-channel currents in cultured rat muscle cells, one-channel NMDA receptor recordings, and mechanically activated PIEZO-channel recordings[1][3][4][5]. The method depends on forming a high-resistance pipette-membrane seal, commonly described as a gigaohm seal, which reduces leak and noise sufficiently to resolve picoampere-scale single-channel currents[2][3]. In the cell-attached configuration, the patch membrane is not ruptured, so cytosolic composition is not directly dialyzed by the pipette solution[3][4].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use a physiological bath solution compatible with the selected cells and a pipette solution chosen for the channel being studied; for ligand-gated channels, agonist or modulator can be included in the pipette solution so it contacts the extracellular face of the membrane patch[4].
• For NMDA receptor one-channel recordings, published examples used HEK293 cells expressing NMDA receptors and native cortical neurons, while PIEZO examples used cell-attached pressure stimulation of mechanosensitive channels[4][5].
• Required instruments are a patch-clamp amplifier, headstage, digitizer or recording interface, micromanipulator, inverted or upright microscope suitable for visualizing cells, glass patch pipettes, pipette puller, bath/reference electrode, vibration isolation, and data-acquisition software; these components support seal formation, voltage or current command delivery, and low-noise current recording from membrane patches[2][4].
• For mechanosensitive-channel recordings, a pressure-clamp or controlled-pressure source is used to apply negative pressure through the recording pipette[4][5].
• For NMDA receptor one-channel recordings, published examples used HEK293 cells expressing NMDA receptors and native cortical neurons, while PIEZO examples used cell-attached pressure stimulation of mechanosensitive channels[4][5].
• Required instruments are a patch-clamp amplifier, headstage, digitizer or recording interface, micromanipulator, inverted or upright microscope suitable for visualizing cells, glass patch pipettes, pipette puller, bath/reference electrode, vibration isolation, and data-acquisition software; these components support seal formation, voltage or current command delivery, and low-noise current recording from membrane patches[2][4].
• For mechanosensitive-channel recordings, a pressure-clamp or controlled-pressure source is used to apply negative pressure through the recording pipette[4][5].
Experimental Procedure
• Prepare healthy adherent cells or neurons suitable for visual patching, and use only cultures or preparations in which the channel of interest is expressed or experimentally introduced; examples include HEK293 cells expressing NMDA receptors, cortical neurons with native NMDA receptors, Neuro2A or HEK293T cells expressing PIEZO channels, and denervated frog muscle or cultured rat muscle cells in classic single-channel studies[1][3][4][5].
• Pull glass patch pipettes and fill them with the selected pipette solution; include the extracellular ligand in the pipette when the goal is to record ligand-gated channels from the extracellular face of the cell-attached patch[4].
• Mount the cell preparation in the bath, connect the pipette electrode to the amplifier headstage, place the reference electrode in the bath, and visually position the pipette near a target cell[2][4].
• Approach the cell with the filled pipette, make contact with the membrane, and apply gentle suction to form the cell-attached seal; retain the membrane patch intact and do not apply the stronger suction or electrical pulse used to rupture the patch for whole-cell recording[2][4].
• Begin recording after seal formation and hold the pipette at the command potential required by the channel and experimental design[2][4].
• For ligand-gated one-channel recordings, acquire long current traces from patches that show one-channel activity so that opening and closing events can be analyzed over time[4].
• For mechanosensitive PIEZO recordings, apply controlled negative pressure through the pipette to stimulate the patch and record pressure-evoked currents[4][5].
• For voltage-activated channels, interpret the patch voltage cautiously because cell-attached voltage-clamp recordings can be distorted by transmembrane voltage changes generated by current flow through activated channels[6].
• Analyze traces by identifying closed and open current levels, measuring single-channel current amplitude, estimating conductance from current-voltage relations when voltage protocols are used, and quantifying open probability, dwell-time distributions, and kinetic behavior[3][7][8].
• Dwell-time histograms can be displayed on logarithmic time axes, and model-based dwell-time fitting can be used when the goal is kinetic modeling of single-channel gating[7][8].
• Use a negative control appropriate to the channel system, such as non-transfected cells for heterologous expression experiments or vector controls in PIEZO experiments, and use a positive control such as cells known to express the target channel or patches showing the expected ligand-, voltage-, or pressure-evoked activity[4][5].
• For cell-attached measurements of voltage-activated ensemble activity, report the limitation that patch current can alter the true transmembrane voltage and distort amplitude or kinetics[6].
• Pull glass patch pipettes and fill them with the selected pipette solution; include the extracellular ligand in the pipette when the goal is to record ligand-gated channels from the extracellular face of the cell-attached patch[4].
• Mount the cell preparation in the bath, connect the pipette electrode to the amplifier headstage, place the reference electrode in the bath, and visually position the pipette near a target cell[2][4].
• Approach the cell with the filled pipette, make contact with the membrane, and apply gentle suction to form the cell-attached seal; retain the membrane patch intact and do not apply the stronger suction or electrical pulse used to rupture the patch for whole-cell recording[2][4].
• Begin recording after seal formation and hold the pipette at the command potential required by the channel and experimental design[2][4].
• For ligand-gated one-channel recordings, acquire long current traces from patches that show one-channel activity so that opening and closing events can be analyzed over time[4].
• For mechanosensitive PIEZO recordings, apply controlled negative pressure through the pipette to stimulate the patch and record pressure-evoked currents[4][5].
• For voltage-activated channels, interpret the patch voltage cautiously because cell-attached voltage-clamp recordings can be distorted by transmembrane voltage changes generated by current flow through activated channels[6].
• Analyze traces by identifying closed and open current levels, measuring single-channel current amplitude, estimating conductance from current-voltage relations when voltage protocols are used, and quantifying open probability, dwell-time distributions, and kinetic behavior[3][7][8].
• Dwell-time histograms can be displayed on logarithmic time axes, and model-based dwell-time fitting can be used when the goal is kinetic modeling of single-channel gating[7][8].
• Use a negative control appropriate to the channel system, such as non-transfected cells for heterologous expression experiments or vector controls in PIEZO experiments, and use a positive control such as cells known to express the target channel or patches showing the expected ligand-, voltage-, or pressure-evoked activity[4][5].
• For cell-attached measurements of voltage-activated ensemble activity, report the limitation that patch current can alter the true transmembrane voltage and distort amplitude or kinetics[6].
Troubleshooting
Problem: No channel openings are observed.
• Possible cause: The patch may not contain an active channel, the target channel may not be expressed, or the necessary stimulus is absent.• Literature-supported solution: Use preparations with verified channel expression, include extracellular ligand in the pipette for ligand-gated channels, or apply controlled negative pressure for mechanosensitive PIEZO recordings[4][5].
Problem: Excess leak or poor resolution prevents single-channel detection.
• Possible cause: The pipette-membrane seal is insufficiently high resistance.• Literature-supported solution: Re-form the cell-attached patch and proceed only after obtaining a high-resistance seal capable of resolving single-channel currents[2][3][4].
Problem: Voltage-activated current amplitudes or kinetics appear distorted.
• Possible cause: In cell-attached voltage clamp, current through activated channels can change the actual transmembrane voltage of the patch.• Literature-supported solution: Interpret voltage-activated ensemble data with this limitation explicitly stated, and avoid treating pipette command voltage as a complete description of patch transmembrane voltage when large patch currents are present[6].
Problem: Dwell-time analysis gives unstable or poorly interpretable kinetic parameters.
• Possible cause: Single-channel events may be idealized or binned in a way that biases dwell-time distributions.• Literature-supported solution: Use logarithmic dwell-time display and model-based fitting methods designed for single-channel dwell-time distributions[7][8].
Références:
- [1]. Neher E, et al. Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature. 1976;260(5554):799-802. [Content Brief]
- [2]. Hamill OP, et al. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981;391(2):85-100. [Content Brief]
- [3]. Sigworth FJ, et al. Single Na+ channel currents observed in cultured rat muscle cells. Nature. 1980;287(5781):447-449. [Content Brief]
- [4]. Maki BA, et al. One-channel cell-attached patch-clamp recording. J Vis Exp. 2014;(88):51629. [Content Brief]
- [5]. Coste B, Mathur J, Schmidt M, Earley TJ, Ranade S, Petrus MJ, et al. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science. 2010;330(6000):55-60. [Content Brief]
- [6]. Williams SR, et al. Errors in the measurement of voltage-activated ion channels in cell-attached patch-clamp recordings. Nat Commun. 2011;2:242. [Content Brief]
- [7]. Sigworth FJ, et al. Data transformations for improved display and fitting of single-channel dwell time histograms. Biophys J. 1987;52(6):1047-1054. [Content Brief]
- [8]. Qin F. Model-based fitting of single-channel dwell-time distributions. Biophys J. 2004;87(3):1657-1671. [Content Brief]
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