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Patch clamp technique was applied to variety of cells for variety of purposes. There are several modifications made for the technique and we can now record not only from the single channel but also from the whole cell membrane. The application of the patch recording technique was expanded and recordings were made recently from brain slice preparations. We have summarized technical aspects and several applications of patch clamp in this short review.
The patch clamp technique affords unparalleled resolution of the detailed properties of ion channel currents. Patch clamping is not difficult and has been used to record single-channel currents from many cell types. The number of artifacts associated with the method appears to be rather small. The main experimental difficulties arise from the need to process large amounts of data. With the development of inexpensive computers and mass storage devices, these problems should be alleviated. The study of membrane excitability is expanding rapidly owing to the introduction of high-resolution patch clamp techniques. The conceptual revolution that will inevitably follow is just beginning.
The use of the patch clamp recording in combination with the technique of the brain slice is the forefront method widely used in the studies of the neurocircuit in vitro. However, few authors in the papers published in domestic journals adopted this method. This paper presents the author's work to modify some details of blind method and the perforated patch clamp method. We used a dissecting microscope (with 40 times object lens) to replace the reverse microscope. As the recording electrode is very important in the experiment, we studied the size and the shape of the electrode tip and used two to three steps to pull the electrode so that the process of polishing of the electrode tip was not necessary and in perforated patch recording the synaptic transmission could be observed at the depolarization of 0-40 mV. The author also showed typical current or voltage waveforms in voltage or current mode respectively for guiding how to obtain the tight-seal whole cell patch. As these modifications have facilitated the acquisition of stable recording and lowered the cost of instrumentation, the methods become more suitable for the use in domestic laboratories.
We describe a method for recording Na+ and K+ currents (INa and IK) from small, voltage-clamped patches of sarcolemma by means of fire-polished glass micropipettes of 7-15 microns tip diameter. Recordings can be made successively from many areas of one fibre. On a given fibre, the amplitudes of INa and IK varied from point to point. Maximum Na+ current densities varied up to three-fold over distances of 10-30 microns, typically between 4 and 12 mA/cm2. K+ currents showed somewhat less lateral variation. Local densities of INa and IK showed no correlation. Apparently the density of Na+ (and, to a lesser extent, K+) channels varies laterally. A contour map of Na+ channel density is constructed for a 20 microns X 90 microns section of sarcolemma. Based on the steepness of lateral gradients in channel density and the estimated survival time of a Na+ channel, it is calculated that at least half of the Na+ channels have a lateral diffusion coefficient of less than 2 X 10(-12) cm2/s. This is three orders of magnitudes less than expected from their molecular size, and suggests that these channels are anchored in the sarcolemma.
We describe a simple and inexpensive experimental chamber that is designed to overcome several problems encountered when doing electrical and optical studies on single cells or on isolated membrane patches. The bath is small enough to fit on the stage of a standard inverted microscope. It includes a novel solution level-detector, the output of which is used to actively control the level of solution in the experimental chamber. A Peltier-effect device is located adjacent to the flow-chamber and heats or cools the inflowing solution. Solutions can be rapidly switched using two electrically actuated microvalves. The attraction of this system is that, with appropriately quiet power supplies, not only is the bath solution-level held at a fixed height, but the temperature of the bathing solution can also be set over a wide temperature range (minimum range is 15 to 45 degrees C), and solutions can be rapidly changed. All of the construction details are supplied as are appropriate electrical circuits. Without modification, the chamber can be used for applications as diverse as fluorescence microscopy of living cells, time-lapse photomicroscopy and single-cell motion detection as well as single cell voltage-clamp and isolated membrane patch-clamp. With simple modification the system can be adapted for use in experiments on multicellular preparations.
Patch clamp techniques were applied to outer mitochondrial membranes of giant mitochondria from mice kept on a cuprizone diet or to vesicles produced by fusing membranes derived from the outer membrane of Neurospora mitochondria. In the negative range of potentials the conductances decreased with increases in the magnitude of voltage, suggesting the closing of channels. Experiments in which mitochondria were treated with the polyanion polymethacrylate maleate styrene (1:2:3) or succinic anhydride suggest that the channels correspond to VDAC. Although sometimes conductance also decreased with increasing potential over a narrow range of positive potentials, more commonly the conductances increased. Although this phenomenon may represent a detachment of the patch, the changes in conductance are reversible, suggesting that they correspond to the formation or the opening of channels.
Patch clamping of myoballs to record single channels was performed to examine the interaction of the anticholinesterase agent pyridostigmine (Pyr) with the acetylcholine (ACh) receptor-ion channel complex. Single ACh channel currents were recorded from tissue-cultured muscle cells of neonatal rats (myoballs). Pyr (50-100 microM) decreased the frequency of channel-opening events activated by ACh, and induced a modified form of the ACh channel currents. Channel conductance was lower in the presence of Pyr, and channel lifetime remained unaltered or only slightly prolonged. In addition, channel openings were frequently interrupted by fast flickers in the presence of Pyr. Higher concentrations (200 microM-1 mM) of the drug induced irregular waves of bursting activity during the initial phase of the application, and, subsequently, significantly reduced the frequency of channel openings. Infrequent channel openings with low conductance were observed in the patch when the micropipette was filled with Pyr alone. These results suggest that, in addition to its anticholinesterase activity, Pyr reacts with the ACh receptor, and both alone or in combination with ACh induces an altered, desensitized species of the nicotinic receptor-ion channel complex.
Nuclear patch clamp is an emerging research field that aims to disclose the electrical phenomena underlying macromolecular transport across the nuclear envelope (NE), its properties as an ion barrier and its function as an intracellular calcium store. The authors combined the patch clamp technique with atomic force microscopy (AFM) to investigate the structure-function relationship of NE. In principle, patch clamp currents, recorded from the NE can indicate the activity of the nuclear pore complexes (NPCs) and/or of ion channels in the two biomembranes that compose the NE. However, the role of the NPCs is still nuclear because the observed NE current in patch clamp experiments is lower than expected from the known density of the NPCs. Therefore, AFM was applied to link patch clamp currents to structure. The membrane patch was excised from the nuclear envelope and, after electrical evaluation, transferred from the patch pipette to a substrate. We could identify the native nuclear membrane patches with AFM at a lateral and a vertical resolution of 3 nm and 0.1 nm, respectively. It was shown that complete NE together with NPCs can be excised from the nucleus after their functional identification in patch clamp experiments. However, we also show that membranes of the endoplasmic reticulum can contaminate the tip of the patch pipette during nuclear patch clamp experiments. This possibility must be considered carefully in nuclear patch clamp experiments.
Patch-clamp performance can be improved without altering the normal headstage configuration described by (Hamill, O. P., A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth, 1981, Pfluegers Arch. Eur. J. Physiol., 391:85-100). The "supercharging" method permits resolution of such fast events as calcium and sodium tail currents. Digital computer modeling and analog electronic simulation were used to identify appropriate shapes for the command voltage and the voltage applied to a capacitor tied to the input of the headstage. The voltage command pulse consists of a step with a brief (5-15 microseconds) rectangular spike on its leading edge. Spike amplitude is a function of the membrane capacitance and the access resistance. The spike drives current through the access resistance and speeds charging of the membrane capacitance, making it possible to complete a voltage step within 5-15 microseconds. Clamping speed is independent of the electrode and feedback resistance over a wide range. The second function of the patch clamp amplifier is current measurement, and good time resolution requires suppression of the capacity transient. This can be accomplished by applying an appropriately shaped voltage to the small capacitor tied to the input of the headstage. Series resistance compensation for ionic current transients does not interfere with supercharging. Although the focus of this paper is on whole cell recording, the supercharging concept may prove useful for single channel and bilayer recording techniques.
Patch-clamp technology has greatly increased our knowledge of plant membrane transport. However, the success of patch clamping crucially relies on establishing a high resistance (G omega) seal between the membrane and the patch-clamp pipette. This can prove problematic in many plant-cell preparations. It is therefore of great importance to develop protocols for protoplast isolation, maintenance and seal formation that improve seal rate. This study investigated whether the pH and the K+ and the Cl(-)concentration of the pipette solution had an effect on the seal formation. High pH and absence of K+ significantly promoted membrane sealing, whereas the concentration of Cl- had no effect. To reap the benefit of seal-promoting pipette solutions and yet retain the option to adjust this solution to experimental requirements, a pipette perfusion apparatus was implemented. The perfusion system was successfully applied in cell-attached patch, excised-patch and whole-cell configurations, using plasma membrane and tonoplast of three different species. The system enables complete solution exchange within minutes and is potentially of great benefit in the study of channel selectivity, the application of (cytoplasmic) channel blockers and the study of primary and secondary transport.
The patch-clamp technique is the state-of-the-art technology for the study of a large class of membrane proteins called ion channels. Ion channels mediate electrical current flow, have crucial roles in cellular physiology, and are important drug targets. However, patch clamping is a laborious process requiring a skilled experimenter and is, therefore, not compatible with the high throughput needed in drug development. The solution for automated and parallel patch-clamp measurements that is provided by microchip technology is presented here.
Patch-clamp amplifiers (PCAs) are commonly used to characterize voltage- and current-clamp responses in the same cell. However, the cell membrane voltage response can be severely distorted by PCAs working in the current-clamp mode. Here we compare the voltage response of pigeon semicircular canal hair cells in situ, recorded with two different PCAs, and with a classic microelectrode bridge amplifier (BA). We found that the voltage response of hair cells recorded with PCAs differed significantly from that recorded with the BA. The true hair cell membrane voltage response to positive current steps was characterized by a strongly damped oscillation, whose frequency and duration depended on hair cell location in the sensory crista ampullaris.
Conventional and automated patch clamp electrophysiology were used to characterise the Na+ current of the SJ-RH30 human rhabdomyosarcoma. In conventional recordings SJ-RH30 cells exhibited a fast activating, fast inactivating Na+ current at potentials positive to -40 mV; in full current-voltage curves maximum current occurred between -20 and -10 mV. Inactivation kinetics at 0 mV were biexponential with time constants of 0.5 and 3.7 ms. Deinactivation at -90 mV also exhibited two kinetic components. Tetrodotoxin (TTX) blocked the Na+ current completely at 1 microM. The NaV 1.4 selective toxin mu-CTx-GIIIB reversibly blocked the Na+ current approximately 60% at 10 microM. Very similar biophysical behaviour was observed in automated patch clamp and conventional recordings. For example, inactivation mid-point was -72+/-2 mV (slope factor 7.2+/-0.2) in automated patch clamp and -74+/-2 mV (slope factor 7.4+/-0.4) with conventional recording. The corresponding values for activation mid-point were -33.2+/-2.4 and -30.3+/-2.7 mV (slope 5.8+/-0.3 and 6.4+/-0.3, respectively). The throughput of the automated method was used to generate additional pharmacological data on inhibition of the Na+ current. TTX inhibited with an IC50 of 23 nM. Mu-CTx-GIIIB also inhibited the channel in a concentration-dependent manner. Inhibition produced by both tetracaine and amitriptyline were shown to be frequency-dependent. Our experiments indicate that the Na+ current of SJ-RH30 cells arises mainly from channels with a phenotype like recombinant NaV 1.4 channels. The suitability of these cells for automated patch clamp suggests they may be useful for higher throughput studies of the interaction of drugs with human skeletal muscle Na+ channels.
The patch clamp method measures membrane currents at very high resolution when a high-resistance 'gigaseal' is established between the glass microelectrode and the cell membrane (Pflugers Arch. 391 (1981) 85; Neuron 8 (1992) 605). Here we describe the first use of the silicone elastomer, poly(dimethylsiloxane) (PDMS), for patch clamp electrodes. PDMS is an attractive material for patch clamp recordings. It has low dielectric loss and can be micromolded (Annu. Rev. Mat. Sci. 28 (1998) 153) into a shape that mimics the tip of the glass micropipette. Also, the surface chemistry of PDMS may be altered to mimic the hydrophilic nature of glass (J. Appl. Polym. Sci. 14 (1970) 2499; Annu. Rev. Mat. Sci. 28 (1998) 153), thereby allowing a high-resistance seal to a cell membrane. We present a planar electrode geometry consisting of a PDMS partition with a small aperture sealed between electrode and bath chambers. We demonstrate that a planar PDMS patch electrode, after oxidation of the elastomeric surface, permits patch clamp recording on Xenopus oocytes. Our results indicate the potential for high-throughput patch clamp recording with a planar array of PDMS electrodes.
Although conventional patch-clamp methods provide high information content, they are labor-intensive and suffer from low throughput and high overall cost. Several approaches for achieving high throughput electrophysiology are under development, among which microchip-based patch-clamp systems uniquely achieve a higher degree of miniaturization, faster perfusion and mixing, and lower reagent cost without losing information content. The goal of this study was to establish a benchmark for our biochip technology with 52 chips tested sequentially. We demonstrate that our microfabrication and processing technology is sufficiently mature to produce a consistent hole size. We further demonstrate high-quality planar whole-cell patch clamping with >75% overall success rate at achieving gigaohm seals, followed by stable whole-cell access lasting at least 15 min with access resistance (Ra) below 15 MOmega and membrane resistance (Rm) above 200 MOmega. These biochips are ideally suited for high throughput compound screening for ion channel targets.
Patch-clamp studies of the yeast Schizosaccharomyces pombe reveal that the plasma membrane contains a voltage-gated channel mildly selective for potassium over sodium, lithium, and chloride. The channel exhibits several conductances with a maximum of 153 pS. The channel gates in the region of physiologically relevant voltages, being closed at hyperpolarizing and open at depolarizing voltages. It is not inhibited by tetraethylammonium, quinine, or quinidine applied from the cytoplasmic side of the membrane; similarly, ATP and stretch have no effect. The frequency of its occurrence in patches implies that about 35 channels of this kind are present in the plasma membrane of a single cell.
Patch-clamping mitoplasts, we have observed a complex pattern of conductance transitions. This report discusses primarily the 45, 120-150, 350, and 1,000 pS transitions.
The introduction of the patch clamp technique less than two decades ago revolutionized the study of cellular physiology by providing a high-resolution method of observing the function of individual ionic channels in a variety of normal and pathological cell types. By the use of variations of the basic recording methodology, cellular function and regulation can be studied at a molecular level by observing currents through individual ionic channels. At a cellular level, processes such as signaling, secretion, and synaptic transmission can be examined. In addition, by combining the information from high-resolution electrophysiological recordings obtained by the patch clamp method with modern molecular biological techniques, further insight can be gained into the gene expression and protein structure of ionic channels. Given the ubiquity and importance of ionic channels, it is not surprising that their study has led to a new understanding of the mechanisms of certain disease processes and has given insight into treatments for these diseases. This review gives an historical perspective of the development of the patch clamp technique and an overview of the methodologies currently in use. Examples are shown to illustrate typical uses of the patch clamp technique with emphasis on the variety of recording configurations available and the advantages and drawbacks of each method.