SEARCH · PubMed Health

Results for “patch clamp”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 37 records · Page 2Linked to original sources

PubMed2005

Computer-aided formation of the whole-cell patch-clamp recording configuration.

The conventional patch-clamp technique requires well-trained experimenter. Few commercial automated patch-clamp systems, designed for drug development, are better suited for large-scale research then for standard electrophysiological experiments. Here we describe a state machine for automated recognition of recording states of the patch-clamp experiment. The principle of the state machine is based on evaluation of the charge carried by membrane current during specific time segments in responses to square wave voltage stimulation. The state machine may serve for generating various sound alerts, signals for automated control of other devices, assistance in micromanipulation, internal pipette pressure control, and holding potential adjustments. Algorithm of the state machine, designed to cover wide variety of cell types, was successfully tested on rat ventricular myocytes.

Algorithms↗

PubMed1994

Ionic currents during action potentials in mammalian skeletal muscle fibers analyzed with loose patch clamp.

The loose patch-clamp technique was applied to analyze transmembrane currents during propagating action potentials in superficial fibers of musculi extensor digitorum longus of the mouse in vitro. Experimentally three components were identified in the transmembrane current: 1) a capacitive, 2) an inward sodium, and 3) an outward potassium current. Other components were negligible. The capacitive current was similar in shape to the first derivative of the intracellularly measured action potential. Tetrodotoxin, tetraethylammonium, and 4-aminopyridine, applied in the pipette, were used to identify the contribution in the current by sodium and potassium ions. With extracellularly applied depolarization steps only a sodium current was observed, not a potassium current. Occasionally found outward currents were artifactual. The behaviour of delayed rectifier potassium channels in muscle fiber membranes is discussed in the light of these unexpected findings. We conclude that potassium channel activity contributing to and measured during action potential generation is in some way inaccessible to loose patch extracellular voltage-clamp stimulation and that loose patch action current recording is a useful noninvasive method to analyze membrane conductances involved in action potential generation.

4-Aminopyridine↗

PubMed2002

Ion channels in small cells and subcellular structures can be studied with a smart patch-clamp system.

We have developed a scanning patch-clamp technique that facilitates single-channel recording from small cells and submicron cellular structures that are inaccessible by conventional methods. The scanning patch-clamp technique combines scanning ion conductance microscopy and patch-clamp recording through a single glass nanopipette probe. In this method the nanopipette is first scanned over a cell surface, using current feedback, to obtain a high-resolution topographic image. This same pipette is then used to make the patch-clamp recording. Because image information is obtained via the patch electrode it can be used to position the pipette onto a cell with nanometer precision. The utility of this technique is demonstrated by obtaining ion channel recordings from the top of epithelial microvilli and openings of cardiomyocyte T-tubules. Furthermore, for the first time we have demonstrated that it is possible to record ion channels from very small cells, such as sperm cells, under physiological conditions as well as record from cellular microstructures such as submicron neuronal processes.

Animals↗

PubMed1997

A ryanodine-sensitive calcium store in ascidian eggs monitored by whole-cell patch-clamp recordings.

Using whole cell patch clamp recordings on unfertilized eggs of the ascidian Ciona intestinalis, we are able to detect ryanodine receptors within the oocytes. Our approach is based on measurements of the voltage-activated inward calcium currents. Two types of Ca2+ currents have been described on the oocyte membrane of Ciona: a low threshold slowly activating current, and a high threshold faster one. We show here that caffeine induces a decrease in the intensity of the Ca2+ currents, when applied either externally or internally from the mouth of a patch pipette. Caffeine application mimics fertilization which transiently decreases the high threshold Ca2+ current density during density during the first meiotic cycle. Ryanodine (> 1 nM) has an effect similar to caffeine. This partial decrease in Ca2+ current density elicited by caffeine or ryanodine is prevented by intracellular application of the calcium chelator BAPTA, then imputable to calcium release. In summary, the depolarization-induced Ca2+ current intensity allows monitoring of an intracellular calcium store which is sensitive to low concentrations of ryanodine in Ciona oocytes. Further identification of a ryanodine receptor was obtained by immunological staining with antibodies against mammalian skeletal muscle ryanodine receptor. Ryanodine receptors were asymmetrically localized in the cortex of Ciona eggs. We discuss the methodological relevance of our patch-clamp approach, in connection with the possible biological role of such a ryanodine receptor in the early stages of development.

Animals↗

PubMed1989

A monolithic patch-clamping amplifier with capacitive feedback.

Patch-clamping is an established method for directly measuring ionic transport through cellular membranes with sufficient resolution to observe open/close transitions of individual channel molecules. This paper describes an alternative technique for patch-clamping which uses a capacitor as the transimpedance element. This approach eliminates bandwidth and saturation limitations experienced with resistive patch-clamping amplifiers. A complete monolithic design featuring an on-chip operational amplifier, a capacitor array with gain-ranging from 30 pF down to 0.03 pF, and reset and gain ranging switches has been fabricated using 5 microns CMOS technology. It is shown that the voltage noise of the CMOS operational amplifier limits the overall noise performance, but that performance competitive with conventional instruments can be achieved over a 10 kHz bandwidth, at least for small input capacitances (less than or equal to 5 pF). Results are presented along with an analysis and comparison of noise performance using both resistive and capacitive elements.

Amplifiers, Electronic↗

PubMed1989

Versatile supplement device with remote control for the control of patch clamp experiments.

A versatile device for a patch-clamp amplifier is described. This device contains: (i) an acoustic indicator to monitor the input resistance of the patch pipette, which is used in search-mode to indicate the formation of seals; (ii) two pulse generators; and (iii) a staircase generator to produce various pulse and voltage step programs; (iv) a low-pass filter which is used to filter the output of the patch clamp amplifier; and (v) a remote control which is used to control the entire patch clamp experiment. This remote control is used to switch between search-, current clamp-, and voltage clamp-mode, to activate the respective stimulus potential programs, and to control the tape recorder. This electronic device can be easily connected to patch clamp amplifiers.

Animals↗

PubMed1996

[Possible applications of the "patch-clamp" method in anesthesiologic research; comment].

The patch clamp technique has gained considerable significance during the past two decades since it was developed. Many mechanisms of cell function have been elucidated by the application of the patch clamp method because it was now possible to demonstrate and investigate directly single ion channels in excitable cell membranes. An important discovery, as far as medicine was concerned, was that various clinically used substances interact directly with ion channels. Local anaesthetics, antiarrhythmics, antidiabetics, muscle relaxants are a few examples. For these reasons, more and more clinical physicians use the patch clamp method for their specific investigations. Collaboration between a clinical institute and a basic research department can often be found, which gives clinical research more background and basic science more practical aspects.

Anesthetics↗

PubMed1994

Lipid-glass adhesion in giga-sealed patch-clamped membranes.

Adhesion between patch-clamped lipid membranes and glass micropipettes is measured by high contrast video imaging of the mechanical response to the application of suction pressure across the patch. The free patch of membrane reversibly alters both its contact angle and radius of curvature on pressure changes. The assumption that an adhesive force between the membrane and the pipette can sustain normal tension up to a maximum Ta at the edge of the free patch accounts for the observed mechanical responses. When the normal component of the pressure-induced membrane tension exceeds Ta membrane at the contact point between the free patch and the lipid-glass interface is pulled away from the pipette wall, resulting in a decreased radius of curvature for the patch and an increased contact angle. Measurements of the membrane radius of curvature as a function of the suction pressure and pipette radius determine line adhesion tensions Ta which range from 0.5 to 4.0 dyn/cm. Similar behavior of patch-clamped cell membranes implies similar adhesion mechanics.

Adhesiveness↗

PubMed2006

A high-performance elastomeric patch clamp chip.

Ion channels play key roles in cell physiology and underlie a broad spectrum of disorders. To this day, the gold standard for studying ion channels is the patch clamp technique. Patch clamping involves careful positioning of a fine-tipped glass micropipette onto the surface of the cell to form a high-resistance (>1 Gohms) seal ("gigaseal"), a procedure that is laborious, vibration-sensitive, and not easily amenable to automation. In addition, the solution inside the pipette cannot be easily exchanged. Recently reported patch clamp chips offer the potential of increased throughput, but to date the overall per-cell performance of most designs has been very low when compared to pipettes, and/or the fabrication process is prohibitively expensive. Here we demonstrate a replica-molded elastomeric patch clamp chip incorporating nanofabricated constrictions, which delivers high-stability gigaseals, with success rates comparable to those of pipettes, using rat basophilic leukemia (RBL) cells. The high stability enables exchanges of both the extracellular and intracellular solution during whole-cell recordings. In a sample of 103 experiments, 66 cells (64%) were successfully immobilized at the patch aperture; 38 cells (58% of immobilized cells, 37% of all cells) were successfully gigasealed; and 25 cells (65% of gigasealed cells, 34% of immobilized cells, 24% of all cells) were successfully perforated for whole-cell access. In the last group of 27 experiments, 79% of the cells could be immobilized, of which 68% could be gigasealed and 46% perforated for whole-cell access, indicating that dexterity is important.

Animals↗

PubMed1995

Correlation of open cell-attached and excised patch clamp techniques.

The excised patch clamp configuration provides a unique technique for some types of single channel analyses, but maintenance of stable, long-lasting preparations may be confounded by rundown and/or rapid loss of seal. Studies were performed on the amiloride-sensitive Na+ channel, located on the apical surface of A6 cells, to determine whether the nystatin-induced open cell-attached patch could serve as an alternative configuration. Compared to excised inside-out patches, stable preparations were achieved more readily with the open cell-attached patch (9% vs. 56% of attempts). In both preparations, the current voltage (I-V) relation was linear, current amplitudes were equal at opposite equivalent clamped voltages, and Erev was zero in symmetrical Na+ solutions, indicating similar Na+ activities on the cytosolic and external surfaces of the patch. Moreover, there was no evidence that nystatin altered channel activity in the patch because slope conductance (3-4pS) and Erev (75 mV), when the bath was perfused with a high K:low Na solution (ENa = 80 mV), were nearly equal in both patch configurations. Our results therefore indicate that the nystatin-induced open cell-attached patch can serve as an alternative approach to the excised inside-out patch when experiments require modulation of univalent ions in the cytosol.

Animals↗

PubMed2006

QPatch: the past, present and future of automated patch clamp.

The QPatch 16 significantly increases throughput for gigaseal patch clamp experiments, making direct measurements in ion channel drug discovery and safety testing feasible. Released to the market in the Autumn of 2004 by Sophion Bioscience, the QPatch originated from work done at NeuroSearch (Denmark) in the early days of automated patch clamp. Today, the QPatch provides many unique features. For example, only the QPatch includes an automated cell preparation station making several hours of unattended operation possible. The 16-channel electrode array, called the QPlate, includes glass-coated microfluidic channels for less compound absorption and, hence, more accurate IC(50) values. The microfluidic pathways also allow for very small amounts of compound used for each experiment ( approximately 5 microl per addition). Only the QPatch has four independent pipetting heads for more efficient liquid handling (especially for ligand-gated ion channel experiments). Patch clamp recordings with the QPatch match the high quality of conventional patch clamp and in some cases the results are even better. For example, only the QPatch includes 100% series resistance compensation for the elimination of false positives due to voltage errors. Finally, the modular QPatch 16 was designed with more channels in mind. The upgrade pathway to 48-channels (the QPatch HT) will be discussed.

Animals↗

PubMed1991

An inexpensive inverted microscope for patch-clamp and other electrophysiological studies at the cellular level.

The popularization of the patch-clamp technique has increased the demand for inverted light microscopes that allow the optimal or almost free movement of patch-clamp pipettes and their support drives. However, commercially available models of inverted microscopes have not been specifically designed for this line of research and, as a consequence, patch-clamp pipette movements are restricted by the small space available between the sample, and the light source and its modulating attachments. This paper provides the details for the construction of a relatively inexpensive inverted microscope that meets the specifications required for patch-clamp and other electrophysiological investigations at the cellular level. The microscope allows the free positioning of the conventional probes for patch-clamp, microelectrode amplifiers, and other micromanipulator probes and attachments. The construction of the microscope is simple and, therefore, since it is relatively inexpensive, the microscope may be easily upgraded in many ways for special purposes, including special optical effects. Finally, although the instrument was developed for patch-clamp and classical electrophysiological studies, it may be used in other types of investigations where freedom of microtool movement is imperative, such as in microsurgery applications.

Electrophysiology↗

PubMed1994

Single-channel inositol 1,4,5-trisphosphate receptor currents revealed by patch clamp of isolated Xenopus oocyte nuclei.

Patch clamp of the outer nuclear membrane of isolated Xenopus oocyte nucleus was used to measure the single-channel properties of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R). The observed channel was activated by IP3, inhibited by heparin, and Ca(2+)-selective, with ion permeabilities PCa:PK:PCl = 8:1:0.05. In symmetric KCl buffer, the channel was ohmic (113 picosiemens in 140 mM KCl) at low channel currents but rectified at higher positive currents. The nuclear IP3R exhibited three conductance substates: a main substate occurring approximately 90% of channel open time, a double substate with twice the main substate conductance and a third substate with half the main substate conductance, which was observed rarely. Channel open probability fluctuated over time and among nuclei. Mean open channel durations of the main and double substates were approximately 5 and 1 ms, respectively. Many channels exhibited periods of closure lasting seconds, and most inactivated permanently within 5 min of IP3 stimulation. These results provide the first characterization of the single-channel properties of the IP3R in its native membrane environment and demonstrate that patch clamp electrophysiology of intact nuclei can be used to directly record currents through the IP3R.

Animals↗

PubMed2006

Improved perfusion conditions for patch-clamp recordings on human erythrocytes.

Various configurations of the patch-clamp method are powerful tools for examining the transport of charged solutes across biological membranes. Originally developed for the study of relatively large cells which adhere to solid surfaces under in vitro culture, these methods have been increasingly applied to small cells or organelles in suspension. Under these conditions, a number of significant technical problems may arise as a result of the smaller geometry. Here, we examined these problems using human erythrocytes infected with the malaria parasite, Plasmodium falciparum, a system where experimental differences and the technical difficulty of erythrocyte patch-clamp have hindered universal agreement on the properties of the induced ion channels. We found that patch-clamp recordings on infected erythrocytes are especially susceptible to artifacts from mechanical perturbations due to solution flow around the cell. To minimize these artifacts, we designed a new perfusion chamber whose geometry allows controlled solution flow around the fragile erythrocyte. Not only were recordings acquired in this chamber significantly less susceptible to perfusion artifacts, but the chamber permitted rapid and reversible application of known inhibitors with negligible mechanical agitation. Electrophysiological recordings then faithfully reproduced several findings made with more traditional methods. The new perfusion chamber should also be useful for patch-clamp recordings on blood cells, protoplasts, and organelles.

Animals↗

PubMed1989

A hybrid patch clamp amplifier.

The current-to-voltage convertor used in patch clamping is analyzed for noise generation and the major noise sources determined. A hybrid patch clamp amplifier design is theoretically analyzed. Here it is shown that by differentiation and recombining of signals the original input signal can be reconstructed. Several circuits of this design are described and their performance compared. The optimal signal detection obtained for a 1 ms current pulse width with a signal-to-noise ratio of 1 is 0.025 pA. In these circuits, high frequency attenuation is readily accomplished with a single control. In addition, compensation for the transients which occur with step control voltages is effectively accomplished. With this circuitry, it is shown that for most patch clamp situations, the minimum pulse width and current which can be detected is determined by the patch clamp seal resistance.

Amplifiers, Electronic↗

PubMed2003

CYTOCENTERING: a novel technique enabling automated cell-by-cell patch clamping with the CYTOPATCH chip.

Automats for patch clamping suspended cells in whole-cell configuration must (1) bring isolated cells in contact with patch contacts, (2) form gigaseals, and (3) establish stable intracellular access that allows for high quality recording of ionic currents. Single openings in planar substrates seem to be intriguing simple solutions for these problems, but due to the low rate of formation of whole-cell configurations we discarded this approach. Single openings are not suited for both attracting cells to the opening by suction and forming gigaseals with subsequent membrane rupture. To settle the three tasks with a mechanical microstructure we developed the socalled CYTOCENTERING technique to apply to suspended cells the same operation sequence as in conventional patch clamping. With this method we immobilized selected cells from a flowing suspension on the tip of a patch pipette by suction with a success rate of 97% and formed gigaseals with a success rate of 68%. Subsequent whole-cell recordings and intracellular staining with Lucifer yellow proved the stable access to the cytoplasm. Currently, a chip with an embedded suction opening in glass surrounding the microstructured contact pipette is under development. The processing of this CYTOPATCH chip is compatible to large-volume production. The CYTOPATCH automat will allow for fully automated, parallel, and asynchronous whole-cell recordings.

Animals↗

PubMed1997

Induced membrane hypo/hyper-mechanosensitivity: a limitation of patch-clamp recording.

Practical limitations of the patch-clamp technique when recording mechanogated membrane ion channels are considered. Mechanical overstimulation of the patch or the cell from excessive suction/pressure protocols induces morphological and functional changes. In particular, the plasma membrane becomes decoupled from the underlying cytoskeleton to form either membrane blebs (cell-attached) or ghosts (whole cell). As a consequence, a membrane ion channel may show either a decrease or an increase in its native mechanosensitivity or even acquire mechanosensitivity. The effect varies with ion channel and cell type and presumably arises because of a disruption of membrane-cytoskeleton interactions. We consider that such disruptions are a pathological consequence of excessive mechanical stress, either during or after seal formation, rather than an immutable consequence of patch-clamp recording. By careful attention to the suction/pressure protocols during sealing and throughout recording, such artifacts can be avoided.

Animals↗

PubMed2006

Open-access microfluidic patch-clamp array with raised lateral cell trapping sites.

A novel open-access microfluidic patch-clamp array chip with lateral cell trapping sites raised above the bottom plane of the chip was developed by combining both a microscale soft-lithography and a macroscale polymer fabrication method. This paper demonstrates the capability of using such an open-access fluidic system for patch-clamp measurements. The surface of the open-access patch-clamp sites prepared by the macroscale hole patterning method of soft-state elastic polydimethylsiloxane (PDMS) is examined; the seal resistances are characterized and correlated with the aperture dimensions. Whole cell patch-clamp measurements are carried out with CHO cells expressing Kv2.1 ion channels. Kv2.1 ion channel blocker (TEA) dosage response is characterized and the binding activity is examined. The results demonstrate that the system is capable of performing whole cell measurements and drug profiling in a more efficient manner than the traditional patch-clamp set-up.

Animals↗