Axon patch clamp equipment molecular devices has many years of expertise in conventional electrophysiology, developing the first amplifier in 1983 under its axon brand. Widely used twoelectrode voltage clamp tevc and patch clamp techniques provide reliable electrophysiological information on the properties of many ion channels. The two agagcl electrodes connect inside the oocyte through 3m kcl solution in glass tubes. Automated twoelectrode voltageclamp electrophysiological assay in xenopus oocytes all experiments were conducted with human nachrs expressed in xenopus oocytes.
Membranepipette interactions underlie delayed voltage. Abstract we report a new technique for improving the efficiency of singlechannel recording in the xenopus oocyte expression system. Optimized procedures for the isolation of single follicles, cytoplasmic rna microinjections, the removal of surrounding cell layers, and protein expression in xenopus oocytes are described. Patch clamp amplifiers from single channels to large macroscopic recordings. Oocytes of the toad xenopus laevis are widely used as an expression system for ion channels, transporters and receptors in drug development until now, the low throughput of electrophysiology has prevented its use for secondary functional screening of drug targets. The electrophysiological patch clamp and two electrode voltage clamp endpoint data of the agonistic effects of the tobacco alkaloids are presented in tables 1 and 2, respectively.
A a schematic illustration of the proposed multichannel chemical sensor using xenopus oocytes cells and a fluidic system that can be connected with a mechanical system. Axoclamp 900a for twoelectrode voltageclamp of xenopus. Apr 10, 2020 the equipment and expertise for heterologous expression in xenopus oocytes and characterization of recombinant ion channels using all patch clamp configurations and two electrode voltage clamps are available. Patch clamp electrophysiology the patch clamp technique is a versatile electrophysiological tool for understanding ion channel behavior. The patch of membrane contained within the electrode tip is then ruptured to allow. Using the axoclamp 900a for twoelectrode voltage clamp of. The quality of voltage clamp recordings obtained with this technique is poor when membrane currents are large and when rapid charging of the membrane is desired. Using the axoclamp 900a for two electrode voltage clamp of xenopus oocytes expressing ion channels t. Improved preparation of xenopus oocytes for patch clamp recording. For experimentalists, the xenopus oocyte brings together in a single cell the capacity to. Single electrode voltage clamp is most often done with patchclamp electrodes 6, which provide a tight seal to the cells membrane and a relatively low resistance.
Automated voltageclamp screening for xenopus oocytes. Highthroughput electrophysiology with xenopus oocytes. P2x electrophysiology and surface trafficking in xenopus oocytes. As a result, removal of the vitelline membrane for patch clamp recording produces microscopic. Every cell expresses ion channels, but the most common cells to study with patchclamp techniques include neurons, muscle fibers, cardiomyocytes, and oocytes overexpressing single ion channels. The two electrode voltage clamp tevc technique is used to study properties of membrane proteins, especially ion channels. An electrophysiological characterization of naturally. Oocytes from xenopus laevis are commonly used as an expression system for ion channel proteins. Recoat the ag recording electrode with agcl before each patch clamping session.
Oocytes of the toad xenopus laevis are widely used as an expression system for ion channels, transporters, and receptors in drug development. May 15, 2006 read fluctuations in xenopus oocytes protein phosphorylation levels during two electrode voltage clamp measurements, journal of neuroscience methods on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Two electrode voltage clamp tevc is a conventional electrophysiological technique used to artificially control the membrane potential v m of large cells to study the properties of electrogenic membrane proteins, especially ion channels. Voltage clamp technique an overview sciencedirect topics. Glass microelectrodes were filled with a cushion of 1 % agarose at their tips to prevent kc1 leakage agarosecushion electrodes. Patch clamp and perfusion techniques for studying ion. Single frames were processed and prepared for publication using adobe. Patch clamp electrophysiology is used to study the electrical properties of excitable cells and ion channels. First, remove the electrode wire from the pipette holder and submerge the tip half of the electrode wire in a vial containing fresh bleach for at least 15 minutes. We investigated the mechanism for proton transport of the m2 protein in xenopus oocytes using a two electrode voltage clamp and in cv1 cells using the whole cell patch clamp technique. This forms a seal, isolating a patch of the membrane to enable the flow of currents across this section of the membrane to be measured. Patch clamp electrophysiology the patchclamp technique is a versatile electrophysiological tool for understanding ion channel behavior. The xenopus oocyte cutopen vaseline gap voltageclamp. Xenopus oocytes as a heterologous expression system for studying ion channels with the patch clamp technique article february 2009 with 760 reads how we measure reads.
Twoelectrode voltageclamp tev of xenopus laevis oocytes is easily applied for the rapid screening of ion channel function, in particular in pharmacological experiments. An amplifier designed for twoelectrode wholecell voltage clamping of large cells xenopus oocytes and cell structures squid axons. To use one of the new benchtop patch clamp systems, you first harvest the cells from a tissueculture dish and wash by pelleting them in a microcentrifuge. Axon instruments and accessories, patchclamp accessories. One of the methods introduced by physiologists is the use of xenopus oocytes for expression of c. Pdf improved preparation of xenopus oocytes for patchclamp. Pdf xenopus oocytes as a heterologous expression system. After a few minutes the cells settle onto the electrode array at the bottom of the well, the computer selects an. Voltage clamp recordings from xenopus oocytes dascal. It was found that this sensor could detect different types of chemicals that have only a slight difference in chemical composition with sensitivity of a few parts per billion in solution. However, both of these methods have drawbacks that prevent the acquisition of reliable data for fast voltagegated sodium channels and other fast activating channels in membranes such as those of xenopus oocytes. Our results indicate the potential for highthroughput patch clamp recording with a planar array of pdms electrodes.
Voltage clamp experiments to record membrane currents from neurons utilize wholecell patch clamp recordings with relatively low resistance microelectrodes 35 m. Currents evoked by ach and other compounds of interest were recorded by using an automated twoelectrode voltageclamp system hiclamp, multichannel systems, reutlingen, germany. Patch clamp recording of ion channels expressed in xenopus. Then you open the lid of the patch clamp box and pipette a few microliters of the cell suspension into the recording well. Here we describe the wholecell two electrode voltage clamp and biotinylationwestern blotting techniques to investigate the functional properties and surface trafficking from p2xexpressing oocytes. They are either bundled with an oocyte clamp amplifier or a more comprehensive two electrode voltage clamp workstation, which provide clamping and amplification of measured signals from glass microelectrodessharp electrodes. Electrical characteristics of biological membranes. We demonstrate that a planar pdms patch electrode, after oxidation of the elastomeric surface, permits patch clamp recording on xenopus oocytes. The most common method for their electrophysiological investigation is the twomicroelectrode voltage clamp technique.
Patch clamp and perfusion techniques for studying ion channels. Patch clamp electrophysiology, voltage clamp, action. Today, molecular devices offers a highly evolved line of microelectrode amplifiers for voltage recording, current clamps and voltage clamps, and patch clamping for ion channel. Aim to study the mutual influence of membrane current induced by receptors with diverse coupling modes in xenopus oocytes. Nevertheless, the low throughput of manually performed. The dualcell voltage clamp technique is a specialized variation of the two electrode voltage clamp, and is only used in the study of gap junction channels. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the african clawed frog, xenopus laevis. Our results indicate the potential for highthroughput patch clamp recording with a planar. Xenopus laevis, oocytes, ion channels, patchclamp, macropatch, insideout, outside. Patch clamp and perfusion techniques to study ion channels. Here we present details of a system in which the twoelectrode voltageclamp technique is used to record electrical signals from xenopus oocytes under hydrostatic oil pressures of up to 60 mpa. Thus, the ability to record wholecell currents from oocytes under high hydrostatic pressure would be very useful. In patch clamp experiments, suction is used to attach a micropipette filled with electrolyte solution to the cell membrane.
Gap junctions are pores that directly link two cells through which ions and small metabolites flow freely. These patches may last for many minutes and are suitable for any electrophysiological study of ion channels expressed in xenopus oocytes, including singlechannel recordings. Two microelectrode voltage clamp tevc of xenopus oocytes introduction the voltage clamp technique is used to measure ionic currents in response to precisely controlled changes in the transmembrane potential of an isolated cell. A voltage recording electrode ev monitors membrane potential. To investigate the mechanism for the delayed activation by voltage of the predominant mechanosensitive ms channel in xenopus oocytes, currents were recorded from oncell and excised patches of membrane with the patch clamp technique and from intact oocytes with the two electrode voltage clamp technique. Two electrode voltage clamp tevc is the most common approach for voltageclamping a large cell and the method ideally suited for cells the size of an oocyte. Since its development by sakmann and neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells.
Read fluctuations in xenopus oocytes protein phosphorylation levels during two electrode voltage clamp measurements, journal of neuroscience methods on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at. We present a planar electrode geometry consisting of a pdms partition with a small aperture sealed between electrode and bath chambers. Two microelectrode voltage clamp tevc of xenopus oocytes introduction the voltage clamp technique is a method that allows ion flow across the cell membrane to be measured as an electric current as the transmembrane potential is held under constant experimental control with a feedback amplifier. An amplifier designed for two electrode wholecell voltage clamping of large cells xenopus oocytes and cell structures squid axons. Aug 20, 2018 using the axoclamp 900a for two electrode voltage clamp of xenopus oocytes expressing ion channels t. The large size of these cells allows for relatively easy expression and recording of activity of exogenous ion channels together with neurotransmitter receptors andor various regulatory proteins using the whole. Improved preparation of xenopus oocytes for patchclamp recording article pdf available in pflugers archiv european journal of physiology 4335. Mechanism for proton conduction of the m2 ion channel of. Xenopus is also a unique system for analyses of genome evolution and whole genome duplication in vertebrates, as different xenopus species form a ploidy series formed by interspecific hybridization. Aug, 2004 the patch images were recorded at 60 frames s.
The two electrode voltage clamp recording technique was used to observe the ligandgated and voltagegated ion channel currents expressed in xenopus laevis oocytes without follicles. Furthermore, xenopus oocytes are a leading system for studies of ion transport and channel physiology. Voltage clamp recordings from xenopus oocytes dascal 2000. These systems provide an option for multiple recordings in parallel or serial in xenopus oocytes, although the development of automated patch clamp systems has mainly focused on mammalian cells using the planararraybased approaches, as discussed above. We report a new technique for improving the efficiency of singlechannel recording in the xenopus oocyte expression system.
A similar strategy was used to assess the agonistic effect of the alkaloids from group a in xenopus oocytes experiments supplementary fig. In addition, a simple method for fast solution changes in electrophysiological experiments with ligandgated ion channels is presented. Researchers use this method most commonly to investigate membrane structures expressed in xenopus oocytes. Axon instruments patchclamp amplifiers molecular devices. Xenopus laevis frog ovarian lobes single oocyte 1 mm diameter injection of crna for your information only. Axoclamp 900a for two electrode voltage clamp of xenopus oocytes expressing ion channels molecular devices. Twoelectrode voltage clamping of expressed ion channels in intact oocytes of the south african clawed frog xenopus laevis has been refined to allow stable, lowresistance electrical access to the cytosol 50800 k. Highthroughput electrophysiology with xenopus oocytes ncbi nih.
Conventional methods of oocyte preparation adequate for two electrode voltage clamp often leave residual adhesions between the vitelline and plasma membranes. Twomicroelectrode voltage clamp of xenopus oocytes. Aug 31, 2010 we have constructed a highly sensitive and selective odorant sensor using xenopus oocytes expressing insect olfactory receptors. Explore the use of axoclamp 900a for two electrode voltage clamp of xenopus oocytes expressing ion channels. Conventional methods of oocyte preparation adequate for twoelectrode voltage clamp. During the preparation of oocytes for patch clamp, the 510 min treatment of stripping solution would detach the vitelline membrane from the plasma membrane, which makes possible stripping the vitelline membrane. Ms channels could be activated by stretch in insideout, oncell, and outsideout patch. This technique is possible only on cells with a large nucleus.
Pdf xenopus oocytes as a heterologous expression system for. Voltage clamp recordings from xenopus oocytes request pdf. To summarize, the mrna of bk channels is injected into xenopus laevis oocytes to express channel proteins on the oocyte membrane. Xenopus oocytes serve as a standard heterologous expression system for the study of cloned ion channels. Pdf patchclamp and perfusion techniques to study ion. Highly sensitive and selective odorant sensor using living. The ideal series resistance of the patch pipette is between 11. The xenopus oocyte expression system is ideal for electrophysiological characterization of voltagedependent and liganddependent ion channels because of its relatively low background of endogenous channels and the large size of the cell. P2x electrophysiology and surface trafficking in xenopus. Patch clamp and perfusion techniques to study ion channels expressed in xenopus oocytes, zhang and cui 2018 describe the use of xenopus oocytes to.
Every cell expresses ion channels, but the most common cells to study with patch clamp techniques include neurons, muscle fibers, cardiomyocytes, and oocytes overexpressing single ion channels. Micromolded pdms planar electrode allows patch clamp. The patchserver is an addon tool for automating a manual patch clamp setup. Improved preparation of xenopus oocytes for patchclamp. The most commonly used approaches for this type of measurement are the twoelectrode voltage clamp tevc 154 in xenopus oocytes and the patchclamp techniques 155. B a schematic view of a xenopus oocyte trapped and inserted by two glass capillary electrodes in the fluidic channel. Patch clamp and perfusion techniques for studying ion channels expressed in xenopus oocytes. Dec 07, 2015 see comment in pubmed commons below pflugers arch. However, conventional tev hardware is not straightforwardly operated by technical personnel because adjustment of the electronics requires considerable practical experience.
Improved preparation of xenopus oocytes for patchclamp recording. Measurements can then be recorded via a powerlab and labchart data acqusition system, which is purchased separately. The trusted patch clamp technique is used in electrophysiological studies of ion channels in tissue sections, individual living cells or patches of cell membrane. The oocyte is the cellular precursor for the development of specialized cells. Using the axoclamp 900a for twoelectrode voltageclamp of xenopus oocytes expressing ion channels writing longterm potentiation and depression protocols and the use of filters in data acquisition and the clampfit application.
Xenopus oocytes and functional characterization by electrophysiological methods. Patch acquisition rates can be over 95% with healthy cells, clean pipettes, and an experienced researcher. Xenopus oocytes serve as a standard heterologous expression system for the study of various ligandgated ion channels including atp p2x receptors. These systems are designed for two electrode, wholecell voltage clamping of large cells such as xenopus oocytes and cell structures such as squid axons. In these experiments we used firststagexenopus oocytes, because their nuclei are clearly identifiable due to the transparency of the cells at this stage. While automated patch clamp systems are useful for screening large amounts of compounds in cell lines that stably express high levels of ion channels, the manual patch clamp technique is still necessary for studying ion channel properties in some research areas and for specific cell types, including primary cells that have mixed cell types and. Patch clamp of the outer nuclear membrane of isolated xenopus oocyte nucleus was used to measure the singlechannel properties of the inositol 1,4,5trisphosphate ip3 receptor ip3r. Whole cell patch clamp recordings from morphologically. The portfolio of amplifiers includes axopatch 200b for ultra lownoise singlechannel recordings, multiclamp 700b for wholecell voltage clamp and highspeed current clamp recordings.
This laboratory course introduces into uptodate electrophysiological research using two electrode voltage clamp tevc. Oocytes of the south african frog xenopus laevis are used widely for the expression of mammalian channels and transporters contributing to numerous discoveries in these fields. Twoelectrode voltage clamp of xenopus oocytes under high. Voltage clamp or current clamp technique is performed in any type of excitable cells, mostly neurons, cardiomyocytes, pancreatic beta. Twoelectrode voltage clamp tevc is a conventional electrophysiological technique used to artificially control the membrane potential. Twoelectrode voltage clamp tevc is a conventional electrophysiological technique used to artificially control the membrane potential v m of large cells to study the properties of electrogenic membrane proteins, especially ion channels. The twoelectrode clamp of oocytes offers a series of advantages over patch clamp recording from macropatchesit is simpler, more stable, allows recording at. Xenopus oocytes are extremely robust cells and will tolerate the use of relatively blunt, lowresistance electrodes for passing current. This twoelectrode voltage clamping tevc amplifier features. Two microelectrode voltage clamp tevc of xenopus oocytes.