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PubMed2004

Ion channel drug discovery and research: the automated Nano-Patch-Clamp technology.

Unlike the genomics revolution, which was largely enabled by a single technological advance (high throughput sequencing), rapid advancement in proteomics will require a broader effort to increase the throughput of a number of key tools for functional analysis of different types of proteins. In the case of ion channels -a class of (membrane) proteins of great physiological importance and potential as drug targets- the lack of adequate assay technologies is felt particularly strongly. The available, indirect, high throughput screening methods for ion channels clearly generate insufficient information. The best technology to study ion channel function and screen for compound interaction is the patch clamp technique, but patch clamping suffers from low throughput, which is not acceptable for drug screening. A first step towards a solution is presented here. The nano patch clamp technology, which is based on a planar, microstructured glass chip, enables automatic whole cell patch clamp measurements. The Port-a-Patch is an automated electrophysiology workstation, which uses planar patch clamp chips. This approach enables high quality and high content ion channel and compound evaluation on a one-cell-at-a-time basis. The presented automation of the patch process and its scalability to an array format are the prerequisites for any higher throughput electrophysiology instruments.

Drug Design↗

PubMed1987

Theory of the kinetic analysis of patch-clamp data.

This paper describes a theory of the kinetic analysis of patch-clamp data. We assume that channel gating is a Markov process that can be described by a model consisting of n kinetic states and n(n - 1) rate constants at each voltage, and that patch-clamp data describe the occupancy of x different conductance levels over time. In general, all the kinetic information in a set of patch-clamp data is found in either two-dimensional dwell time histograms describing the frequency of observation of sequential dwell times of durations tau 1 and tau 2 (Fredkin, D. R., M. Montal, and J. A. Rice, 1985, Proceedings of the Berkeley Conference in Honor of Jerzy Neyman and Jack Kiefer, vol. 1, 269-289) or in three-point joint probability functions describing the probability that a channel is in a given conductance at time t, and at time t + tau 1, and at time t + tau 1 + tau 2. For the special case of channels with a single open state plus multiple closed states, one-dimensional analyses provide all of the kinetic information. Stationary patch-clamp data have information that can be used to determine H rate constants, where H = n(n - 1) - G and G is the number of intraconductance rate constants. Thus, to calculate H rate constants, G rate constants must be fixed. In general there are multiple sets of G rate constants that can be fixed to allow the calculation of H rate constants although not every set of G rate constants will work. Arbitrary assignment of the G intraconductance rate constants equal to zero always provides a solution and the calculation of H rate constants. Nonstationary patch-clamp data have information for the determination of H rate constants at a reference voltage plus n(n - 1) rate constants at all test voltages. Thus, nonstationary data have extra information about the voltage dependencies of rate constants that can be used to rule out kinetic models that cannot be disqualified on the basis of stationary data.

Electric Conductivity↗

PubMed1986

Epithelial sodium channels: characterization by using the patch-clamp technique.

The patch-clamp technique was used to resolve currents through individual Na-selective ion channels in the apical membrane of the rat cortical collecting tubule. The channels had a single unit conductance of 5 pS under control conditions (cell-attached patches, room temperature, 140 mM NaCl in the pipette). They appeared to be highly selective for Na, as K conduction through them was not measurable in inside-out patches. The channels underwent spontaneous transitions between open and closed states, both states being long-lived. At physiological temperature (37C) the conductance increased to 9 pS and the spontaneous transitions became more rapid. In the presence of amiloride on the luminal side of the membrane, the channel flickered rapidly between open and blocked states. The size of the current transitions did not change. This channel activity was observed only in rats that were fed a low-Na diet to elevate aldosterone secretion. In addition to mineralocorticoid control, the activity of the channels in inside-out patches were modulated by the pH on the cytoplasmic side of the membrane. Alkalinization from pH 6.4 to 7.4 increased the probability of channels' being open by eightfold. Changes in Ca concentration on the cytoplasmic side of the membrane did not directly affect the channels. However, addition of ionomycin, a Ca ionophore, to the bath resulted in a decrease in channel activity measured in cell-attached patches. This suggests that high cytoplasmic Ca may indirectly down-regulate Na channels in this tissue.

Amiloride↗

PubMed2002

Patch clamp electrophysiology in nodose ganglia of adult rat.

The patch clamp technique is widely utilized for studying the electrophysiological properties of enzymatically isolated sensory neurons. Unfortunately, dissociation of the ganglion severs the afferent fibers. As a result, isolated neurons can only be broadly categorized according to somatic action potential waveforms, ion channel subtypes, chemical sensitivities and cell diameter. Such restricted classifications contrast with the continuum of conduction velocities (CVs), discharge patterns, sensory modalities and functional properties of visceral and spinal afferents. Previous reports of patch clamp recordings using intact ganglion have been limited to young animal preparations. This raises concerns regarding postnatal development and impedes the use of chronic models of disease or injury, which often necessitate the use of a more mature animal preparation. Here, we present a methodology for preparing nodose ganglion from adult rat (250-400 g) for study using the patch clamp technique. Successful whole cell recordings were obtained from approximately 50% of the cells selected for study, the majority of which had intact afferent fibers. Measures of somatic discharge and afferent fiber CV at both room and physiological temperatures were consistent with investigations using sharp microelectrodes. Voltage clamp recordings of whole cell Na(+), Ca(2+) and K(+) ion channel currents were comparable to those obtained using isolated neuron preparations. The ability to classify voltage- and ligand-gated ion channel type with afferent fiber CV in an adult preparation adds a valuable new dimension to cellular investigations of the diverse functional and chemical properties of the peripheral afferent nervous system.

Animals↗

PubMed2002

A method of patch clamp recording in hyperbaric oxygen.

A method of patch clamp recording in hyperbaric oxygen is described which is derived from a method for patch clamping at high hydrostatic pressure. Excised patches equilibrate with hyperbaric oxygen within 10 minutes. Experiments with BK channels expressed in HEK 293 cells showed that 1 MPa O2 caused an increase in the ionic permeability of the patch, an effect accelerated in the presence of ferrous ions. Seal resistance was unaffected. Small negative currents, (VH - 40 to - 65 mV, symmetrical KCl solutions), apparent in skewed all-points amplitude histograms, reduced the signal to noise ratio. In some patches the BK channel activity was inhibited and in others it persisted with the currents becoming irresolvable in the increased noise.

Cell Line↗

PubMed1992

[Patch-clamp technique].

We first describe how to set up the system for patch-clamp recording and carry out the experiments with 4 different modes of conventional patch-clamp technique: cell-attached, whole-cell, inside-out and outside-out configurations. Thereafter, we mention about an improved 'nystatin perforated' patch-clamp technique which dissolves the fault of conventional patch-clamp techniques except the cell-attached mode.

Animals↗

PubMed2005

[Advancement of patch-clamp technique and its application in drug high-throughput screening].

The patch-clamp technique, a dominant technique in cellular electrophysiology, is always being regarded as the gold standard for ion channel research. Application of the patch-clamp technique can demonstrate the existences of ion channels and provide valuable information for ion channels, including their electrophysiological properties, molecular structures and the mechanism of drug action. Genomics and proteomics research has showed that the development of drugs for ion channel target would be very promising in future. In recent years, numerous improvements have been achieved, which will facilitate the application of patch clamp technique in drug high-throughput screening. These techniques will break through the bottleneck in the development of drugs aiming at ion channels. New advancements in patch-clamp technique and application in drug high-throughput screening are reviewed in the paper.

Animals↗

PubMed1995

Characterization of mechanosensitive channels in Escherichia coli cytoplasmic membrane by whole-cell patch clamp recording.

Whole-cell patch clamp recordings were done on giant protoplasts of Escherichia coli. The pressure sensitivity of the protoplasts was studied. Two different unit conductance mechanosensitive channels, 1100 +/- 25 pS and 350 +/- 14 pS in 400 mM symmetric KCl solution, were observed upon either applying positive pressure to the interior of the cells or down shocking the cells osmotically. The 1100 pS conductance channel discriminated poorly among the monovalent ions tested and it was permeable to Ca2+ and glutamate-. Both of the two channels were sensitive to the osmotic gradient across the membrane; the unit conductances of the channels remained constant while the mean current of the cell was increased by increasing the osmotic gradient. Both of the channels were voltage sensitive. Voltage-ramp results showed that the pressure sensitivity of protoplasts was voltage dependent: there were more channels active upon depolarization than hyperpolarization. The mechanosensitive channels were reversibly blocked by gadolinium ion. Also they could reversibly be inhibited by protons. Mutations in two of the potassium efflux systems, KefB and KefC, did not affect the channel activity, while a null mutation in the gene for KefA changed the channel activity significantly. This indicates a potential modulation of these channels by KefA.

Antiporters↗

PubMed2006

A novel method for patch-clamp automation.

An increasing demand of the pharmaceutical industry for automated electrophysiological stations for ion channel drug discovery has recently resulted in the development of several commercial platforms for secondary and safety screening of ion channel modulators. These commercial systems have demonstrated an enhanced throughput, however, often at the expense of some quality-sensitive aspects of traditional patch-clamp recordings. To improve data quality and content, we have developed a patch-clamp robot that fully automates manual patch-clamp recordings, including patch pipette handling, gigaseal formation, obtaining whole-cell or perforated-cell configuration, drug application, and data acquisition. Utilization of glass micropipettes results in high-quality electrophysiological recordings with an overall success rate of about 30% in perforated-cell mode. A fast drug application system with low volume requirements (1-1.5 ml) allows the study of ligand-gated ion channels on a millisecond scale. As proof-of-concept, we present two assays developed for voltage-gated human ether-a-go-go-related and ligand-gated alpha(7) nicotinic receptor ion channels. The system throughput was a single concentration-response curve every 30-40 min or 12-17 6-point concentration-response curves daily, representing a significant improvement of typical manual patch-clamp throughput. This system represents an efficient method for patch-clamp automation without the need for a complex and expensive electrophysiological set-up for cell visualization.

Animals↗

PubMed1989

The slow inward calcium current is responsible for a part of the contraction of patch-clamped rat myoballs.

The slow inward calcium current and the contractile response were simultaneously recorded in voltage clamped (whole cell patch clamp recording) rat myoballs in primary culture. The shape of the contraction(T)/potential(V) relationship and the application of the inorganic calcium channel blocker cadmium (1.5 mM), which suppresses a part of the contractile activity, demonstrate the existence of two components of contraction. One of them is related to the slow calcium current.

Animals↗

PubMed2000

LabPatch, an acquisition and analysis program for patch-clamp electrophysiology.

An acquisition and analysis program, "LabPatch," has been developed for use in patch-clamp research. LabPatch controls any patch-clamp amplifier, acquires and records data, runs voltage protocols, plots and analyzes data, and connects to spreadsheet and database programs. Controls within LabPatch are grouped by function on one screen, much like an oscilloscope front panel. The software is mouse driven, so that the user need only point and click. Finally, the ability to copy data to other programs running in Windows 95/98, and the ability to keep track of experiments using a database, make LabPatch extremely versatile. The system requirements include Windows 95/98, at least a 100-MHz processor and 16 MB RAM, a data acquisition card, digital-to-analog converter, and a patch-clamp amplifier. LabPatch is available free of charge at http://www.fhs.mcmaster.ca/huizinga/.

Computer Graphics↗

PubMed1999

[The advances of the research work on lens cells using patch clamp technique].

The study of lens cells by patch clamp technique began in the early 90's. It shows that the membrane of lens cells possess different types of ionic channels. These channels are responsible for the modulation of the hydration state and transparency of lens. Several patch clamp configurations have been used to study the activity of ionic channels. The resting potential and capacitance as well as resistance of different lens cells were also measured.

Aerospace Medicine↗

PubMed1992

Carbachol induces K+, Cl-, and nonselective cation conductances in T84 cells: a perforated patch-clamp study.

We used the perforated patch-clamp technique to examine cell membrane ionic conductances in isolated cells of the human colonic secretory cell line, T84, during exposure to the muscarinic agonist carbachol. Carbachol (100 microM) induced both outward and inward currents when the patch pipette contained a normal intracellular-like solution, the bath contained a normal extracellular-like solution, and the cells were intermittently voltage clamped between K+ and Cl- equilibrium potentials. The outward current was identified as a K+ current that averaged 483 +/- 95 pA, while the inward current averaged 152 +/- 29 pA (n = 15). The outward and inward currents oscillated with a synchronous frequency of 0.036 +/- 0.006 Hz; however, the onset of the K+ current occurred an average of 457 +/- 72 ms before the onset of the inward current. When the pipette contained a high-NaCl solution, the bath contained a Na(+)-gluconate solution, and the cells were intermittently voltage clamped between Cl- and Na+ equilibrium potentials, carbachol induced both Cl- and nonselective cation currents. The Cl- current averaged 455 +/- 73 pA, while the nonselective cation current, averaged 336 +/- 54 pA (n = 14). No difference was observed in the onset of these two currents. These results indicate that carbachol induces three separate ionic conductances in T84 cells. We used the whole cell patch-clamp technique in a previous study of these cells [D. C. Devor, S. M. Simasko, and M. E. Duffey. Am. J. Physiol. 258 (Cell Physiol. 27): C318-C326, 1990] and found that carbachol induced only an oscillating membrane K+ conductance. Thus some unidentified component of the carbachol-sensitive signal transduction pathway is diffusible and may be lost during whole cell patch clamping.

Amiloride↗

PubMed1986

Patch-clamp technique in renal physiology.

The patch-clamp technique of Neher and Sakmann and their colleagues has been widely used over the last 5 years to investigate ion channels in excitable tissues. More recently, it has become useful as a tool to study channels involved in transepithelial ion transport. In this review, I briefly cover the basic concepts behind the patch-clamp technique and the kinds of information that can be obtained with it. I then summarize the applications of the technique to renal tissues and describe some of the channel types that have been observed to date in epithelia.

Animals↗

PubMed2004

Dynamic ion channel activation scheduling in patch clamp on a chip.

In 2002, Fertig et al. made a remarkable invention: the first successful demonstration of a patch clamp on a chip--a planar quartz-based biological chip that contains up to several hundred ion channels. This patch-clamp chip can be used in massively parallel screens for ion channel activity, thereby providing a high-throughput screening tool for drug discovery efforts. In this paper, we propose computationally efficient dynamic stochastic scheduling algorithms for activating individual ion channels in the patch-clamp chip. By formulating the ion channel activation scheduling problem as a partially observed Markov decision process with a multiarmed bandit structure, near-optimal dynamic scheduling for activation of the individual channels is achieved to optimize the information gained from the patch-clamp chip. Numerical examples using state-of-the-art algorithms developed recently in artificial intelligence and operations research are presented to illustrate these dynamic ion channel (macromolecule) activation scheduling algorithms.

Algorithms↗

PubMed2002

[Application of rat tail collagen in patch clamp experiment with vestibular hair cells].

OBJECTIVE: To study the feasibility of application of rat-tail collagen in patch clamp research in vestibular hair cells. METHODS: The effect of self-made rat-tail collagen on promoting adhesion of vestibular hair cells in whole cell patch clamp experiment was observed. RESULTS: A seal was hard to be formed when the vestibular hair cells suspended among the external solution without rat-tail collagen. However, when the vestibular hair cells were firmly adhesive to the bottom of the recording chamber with rat-tail collagen, a seal can be formed easily, which fitted to the long-term observation and recording. The effect of rat-tail collagen on adhesive to vestibular hair cells is obvious. CONCLUSION: Rat-tail collagen could facilitate the vestibular hair cells to adhesive with the bottom of the recording chamber, which is helpful for long-term patch clamp research, and the collagen is considered as an optimal adhesive reagent to vestibular hair cells for patch clamp research.

Animals↗

PubMed2006

[A data interface based on USB bus technology for full auto patch-clamp system].

A USB bus based data interface technology for full auto Patch-Clamp system is discussed in the article. The main controller is CY2131QC (Cypress) and the logic controller is EPM3256A (Altera). Optocouplers are used to get rid of the noise from the interface. It makes the installation of the Patch-Clamp system easier by using the USB bus, and is suitable for the new generation of the Patch-Clamp system with a high speed of 1M bytes/s.

Computer-Aided Design↗

PubMed2006

Population patch clamp improves data consistency and success rates in the measurement of ionic currents.

Present whole-cell patch-clamp methodology has only moderate consistency and throughput, rendering impractical functional measurements on large numbers of ion channel ligands or on large numbers of unknown or mutant channel genes. In the population patch clamp (PPC) described herein, a single voltage-clamp amplifier sums the whole-cell currents from multiple cells at once, each sealed to a separate aperture in a planar substrate well. The resulting ensemble currents are more consistent from well to well, and the success rate for each recording attempt is >95%. The PPC was implemented by modifying the PatchPlate substrate and amplifiers in the IonWorks patch-clamp instrument. The increased data consistency and likelihood of a successful recording in each well, combined with 384-well measurements in parallel, allow the direct electrophysiological recording of thousands of ensemble ionic currents per day. Therapeutic groups in drug discovery programs require this order of throughput to screen directed compound libraries against ion channel targets. The potential for studying the function of large numbers of ion channel mutants may be realized with the technique. The procedure incorporates subtraction methods that correct for expected distortions and also reliably produces data that agree with previous patch-clamp studies.

4-Aminopyridine↗