Patch‐Clamp Technique

Abstract

The patch‐clamp technique enables the recording of bioelectrical signals in excitable and nonexcitable cells. At the molecular level, tight‐seal, high resolution current recording from small membrane patches (∼10 μm2) allows real time monitoring of conformational transitions in single membrane channel proteins as they are gated open (or closed) by specific stimuli, including changes in membrane potential, membrane tension, and specific chemicals/neurotransmitters. Cell‐free membrane patch recording allows characterisation of channels modulated by intracellular messengers (e.g., Ca2+, nucleotides, G‐proteins and phospholipids). Tight‐seal whole‐cell recording can monitor action and synaptic potentials/currents generated by many channels. Furthermore, by using multiple patch pipettes to record at spatially and functionally distinct regions of a cell one can measure precisely the initiation and spread of potential within geometrically complex cells.

Key Concepts:

  • Patch pipette contact with a cell membrane followed by applied suction often results in an abrupt, all‐or‐none, tight seal of high electrical resistance (10–200 GΩ).

  • The remarkable features of the GΩ seal including its abrupt nature, mechanical strength and pH dependence are most consistent with a water‐based mechanism of adhesion in which confined interfacial structured water ‘glues’ the glass and membrane surfaces tightly together.

  • Patch‐clamp recording allows direct measurement of the ionic currents through individual membrane protein ion‐selective channels.

  • With its high temporal (submillisecond) and current (subpicoampere) resolution, patch recording provides the unique opportunity to follow in real time the conformational transitions of single membrane channel proteins as they switch between different discrete conductance states.

  • Patch‐clamp recording also allows the experimental control of the membrane patch potential via voltage‐clamp, and the membrane patch tension via pressure‐clamp, thereby permitting the characterisation of voltage‐ and mechanically gated membrane ion channels, respectively.

  • Single channel patch recording provides the gold standard for assay/identification of purified membrane protein channels reconstituted into liposomes and recombinant channels heterologously expressed in Xenopus oocytes or mammalian cell lines.

  • Cell‐attached patch recording in the current‐clamp mode can be used to monitor noninvasively dynamic changes in the cell's membrane potential (e.g. during synaptic transmission), although in the voltage‐clamp mode it can be used to record and modify action potential firing patterns.

  • Cell‐free patches, including inside‐out and outside‐out configurations, provide the unique opportunity to manipulate the ionic and biochemical environments at both membrane faces to characterise channel ion selectivity/conductance mechanisms and channel gating mechanisms mediated by specific intracellular 2nd messengers including Ca2+, nucleotides, proteins and phospholipids.

  • The ability to selectively record from specific subcellular regions of an individual cell (e.g. sensory receptor specialisations) has demonstrated the heterogeneous distribution of channels including both voltage‐ and mechanically gated channels.

  • Tight‐seal, whole‐cell recording can be used to monitor whole cell currents and potentials in cells too small (<10 μm in diameter) to be studied by sharp microelectrodes (e.g. blood cells and cerebellar granule cells) while at the same time dialyzing the cell with specific biochemicals or labelling dyes.

  • Using multiple patch pipettes to record whole cell potentials from spatially separate and functionally distinct cellular regions (e.g. dendrites, soma and the axonal hillock) one can measure with precise timing the initiation and forward and back propagation of action potentials (i.e. from the spike initiation zone) within geometrically complex cells.

  • Patch‐clamp recording by revealing channel expression in cells from diverse tissues including epithelia, endothelia, glia, blood and most recently stem cells has provided insight into the diverse roles channels play in various physiological and developmental processes.

  • Patch recordings from pathological cells/tissues have revealed the role channels play in many disease states, ‘channelopathies’, including diabetes, muscular dystrophy, cystic fibrosis, cardiac arrhythmias and cancer.

Keywords: patch clamp; whole cell currents and potentials; tight seal; single membrane channel; whole cell recording; pressure clamp; reconstituted channels; mechanosensitive channels; inside‐out patch; outside-out patch; membrane delimited coupling

Figure 1.

Patch‐clamp technique. (a) The schematic shows a patch pipette pressed up against a cell before suction is applied to the inside of the pipette to promote tight sealing. After sealing most of the current generated in the patch will flow up into the pipette to be measured by the patch‐clamp circuitry involving the current‐to‐voltage converter in the head stage in which the feedback resistor (Rf) determines the voltage into current conversion gain according to Voutput=Rfip+Vref, where Voutput represents the output voltage, ip represents the patch current and Vref represents the reference voltage set by the experimenter. (b) Specific resistances that contribute to the pipette tip resistance (Rtip) commonly used to estimate tight seal resistance in which the test pulse flows to ground through the three series resistors Rpatch, Rseal and Rcell so that Rtip=(1/Rseal+1/(Rpatch+Rcell))−1 assuming Relec is relatively small so that it can be ignored (see Perkins ()).

Figure 2.

The time course of tight seal formation on frog red blood cells (RBCs). Patch current recording of sealing on three different frog (Rana pipiens) RBCs. In each case, a 0.1 mV pulse was applied to the pipette to monitor pipette resistance. The three trials illustrate that sealing can occur with (a) brief (1 s) suction, (b) no suction with positive pressure or (c) prolonged (10 s) suction. The differences may be explained in part by the difference pipette solutions used ((a) 90 mM NaCl, (b) 70 mM NaCl and (c) 100 mM KCl). Reproduced from Hamill (). © Plenum Press.

Figure 3.

The membrane patch changes morphology during repetitive mechanical stimuli. (a–d) show video images of a cell‐attached patch at different times after formation of the tight seal. (e–f) show the current response at the time of images (a) and (d), respectively. Between each image, brief suction pressure/suction protocols pulses were applied causing the membrane to progressively move up the pipette and a clear space developed between the membrane and the underlying cytoplasmic structures. Accompanying this membrane blebbing the mechanosensitive current was reduced in size and the initial fast kinetic phase was abolished. Modified from Zhang et al. ().

Figure 4.

Streaming currents measured across the tight seal. Current responses to suction pulses applied to a cell‐attached patch tightly sealed on a PC3 cell that expressed no MS channel activity. The presence of streaming current indicates electrolyte and mobile ions are present within the confined space of the seal.

Figure 5.

Different physical mechanisms to explain tight seal formation. Cartoons illustrating the different mechanisms of adhesion proposed to underlie the tight GΩ seal formed between glass and membrane. (a) A model that assumes that the glass and membrane surfaces make atomic contact so that electrodynamic Van der Waals forces overcome any repulsive electrostatic forces. In this model all electrolytes are excluded from the seal space so that Rseal should approach infinity, show little of no ionic/pH dependence and display high resistance to patch movement. (b) A model based on the idea that dielectric surfaces of opposite polarity cause a complementary alignment of water molecules that reach across the space to glue the surfaces together. In this model, cations like K+ and Na+ are depleted from the nanospace because they disrupt the water structure, whereas H3O+ ions, because they can form hydrogen bonds, are accommodated (not shown). (c) Like‐likes‐like attraction is proposed to occur because interfacial water at the hydrophilic surfaces takes on an ice‐like structure that is negatively polarised, excludes solutes, extends out as far as 100 μm from the surface, and autocatalyses the production of H3O+ at the edge of the interfacial water. It is the intervening H3O+ that is proposed to produce the like‐likes‐like attraction.

Figure 6.

High‐resolution video images of a tightly sealed membrane patch and the patch current during brief steps of suction and pressure. (a) and (b), images of the patch before, during and after applying steps of suction (a) and pressure (b). (c) and (d), the patch current recorded in the same patch imaged in (a) and (b), respectively, in response to suction and pressure steps. Modified from Zhang and Hamill ().

Figure 7.

Schematic representation of the procedures used to form the different patch‐clamp configurations. To obtain the cell‐attached patch configuration the pipette tip is pressed against the cell and suction is applied to the pipette interior (top figures). To form the inside‐out patch the pipette is pulled away from the cell leaving a vesicle tightly sealed in the tip. The pipette tip is then briefly passed through the solution–air interface, which acts to disrupt the outer membrane of the vesicle (right hand figures). To form the outside‐out patch, stronger suction is applied to the cell‐attached patch until it ruptures gaining access to the inside of the cell. The pipette is then pulled away from the cell leaving the outer membrane face facing out and exposed to the external bath solution. Modified from Hamill et al. ().

Figure 8.

Patch‐clamp recording can be used to identify different types of coupling between receptors and channels. Each panel shows a cell‐attached patch with a specific type of coupling between the channel and receptor. (a) Intrinsic coupling in which the receptor and channel are part of the same molecule. (b) 2nd messenger coupling in which activation of the receptor results in synthesis or release of a soluble 2nd messenger that can diffuse significant distance to activate the channel. (c) Membrane delimited coupling in which a structural component of the receptor dissociates from the receptor and diffuses within the membrane to activate channels in proximity. (d) Conformational coupling in which a structural change in the receptor is directly transmitted to the channel molecule. In (a), (b) and (c) inclusion of agonist in the pipette activates the channel but bath‐applied agonist only activates the channel in (b) because in this case the soluble 2nd messenger is able to diffuse into the patch and reach the channel. In (d) agonist included in either the pipette or the bathing solution may fail to activate the channel in the patch because the process of tight sealing physically disrupts the coupling between receptor and channel.

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Ashcroft FM (2000) Ion channels and disease: Channelopathies. Amsterdam, Holland: Elsevier Science.

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Sakmann B and Neher E (eds) (1995) Single‐Channel Recording, 2nd edn. New York: Plenum Press.

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Hamill, Owen P(Apr 2014) Patch‐Clamp Technique. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0003382.pub2]