Patch‐Clamp Technique

Abstract

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 μm 2 ) 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., Ca 2+ , 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 2 nd messengers including Ca 2+ , 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.