Patch clamp traditionally uses a glass pipette, with an open tip diameter of about one micrometre, and is made such that the tip forms a smooth surfaced circle, rather than a. How to use channelmax and patchmax for cultured cells. Unfortunately 99% of the time i dont see any channel activity even at. The patchserver is an addon tool for manual patch clamp. Functional overview of series 20 chambers warner instruments. Although channelmax and patchmax are originally designed for oocyte patch clamp, they can be used for cultured cells patch clamp. Schematic representations of four patch clamp configurations. Lipid bilayer voltage clamp with reconstituted na channels. Prior to patch clamp measurements, the vitelline membrane, surrounding the xenopus oocyte, was manually removed with a pair of forceps to get access to the plasma membrane. Cell attached and cell free patch clamp techniques have become the methods of choice for the analysis of the functional properties and subcellular distribution of voltageactivated ion channels. Defolliculated oocytes were manually devitellinized in nd96 medium immediately before patchclamp experiments.
Pipette solution was alternately perfused with mg70 indicated with open bar and ca70 solid bar. Patch clamp amplifiers, auto planer oocyte scitech. Close the electronic valve connected to the tubing of waste collector and open the. The cutopen oocyte vaseline gap voltage clamp technique allows for rapid resolution of data, low noise, increased control over internal solution and external solution composition, and stable recordings over relatively long protocols 19. These channels are seen when suction is applied to the patch pipette, but not in the absence of suction or during voltage steps. Alternatively, investigators can pull a patch of membrane away from the cell and evaluate currents through single channels via the insideout or outsideout patch clamp technique.
The xenopus laevis oocyte is a widely used system for heterologous expression of exogenous ion channel proteins 1, 2. Which patch clamp configuration is formed by retracting a pipette that is in the whole cell arrangement. Ground lead cables ground lead cable and solder pin cup packs for use with your model cells and patch clamp recordings. The membrane attached to the pipette follows the retreating pipette, and a. The female germ cells produce a primordial germ cell pgc, which then undergoes mitosis, forming oogonia.
Patchclamp techniques for ion channels in xenopus oocytes. The extrapolated reversal potential indicated that the channels are chlorideselective. Oocyte patch clamping was performed by standard techniques. For patchclamp you will anyway use excised most probably, outsideout patch.
Ionic current of bk channels is recorded using patch clamp techniques. Oocyte is confined in a cone shaped well to a fixed position. Xenopus laevis, oocytes, ion channels, patchclamp, macropatch, insideout. In patch clamp experiments, suction is used to attach a micropipette filled with electrolyte solution to the cell membrane. Patch clamp electrophysiology, voltage clamp, action. Wholecell recording is achieved by destroying the membrane patch using suction. Axon instruments and accessories, patchclamp accessories. Enac whole cell currents were insensitive to mechanical cell swelling caused by direct injection of 90 or 180 nl of 100mm kcl. The success of performing patch clamp experiments on in situ nuclei was dependent on a specific preparation. In other words, it is an immature ovum, or egg cell. Enac wholecell currents were insensitive to mechanical cell swelling. Perfusion chamber i this perfusion chamber is designed for oocyte twoelectrode voltage clamp and patch clamp. Epithelial sodium channel inhibition by ampactivated.
However, once a patch was formed on an activated cell, i. How i can attach oocytes on plate for doing patch clamp. Voltage ramps were applied periodically to obtain the iv curves shown in b. Limitations of cell attached patch clamp recording from highresistance neurons the present results show that the generalized reputation of cell attached recordings as being noninvasive is illdeserved, because such recordings can significantly influence the firing rate of highresistance neurons such as cerebellar mlis. These advantages set this technique apart from the standard twoelectrode voltage clamp and patch clamp. Patch clamp techniques for single channel and wholecell. Advanced circuit design techniques using field programmable gate arrays eliminated noisy microprocessors.
Excision of cellattached patches is achieved by quickly removing. Cellattached patches are formed while establishing the gigaohm seal in patchclamp recording techniques. Patch clamp measurements were made in the cellattached configuration. Gigaseal patch clamp cell attached and excised patches.
After gigaseal formation, the membrane patch is ruptured with negative pressure or a voltage pulse to create a lowresistance access into the intracellular compartment fig. Measuring the firing rate of highresistance neurons with. In the cell attached patch clamp configuration, the currentvoltage curve trace c. The electrode is tightly sealed to the patch of the membrane and the cell remains intact.
This technique is possible only on cells with a large nucleus. Applications include patch clamp, oocyte clamp, and other physiological measurements on cell cultures and tissue preparations. Pdf xenopus oocytes as a heterologous expression system for. Model cells are available for oocytes, twoelectrode voltage clamp discontinuous singleelectrode voltage clamp, whole cell singlechannel patch clamp, and bilayer conditions. Measurements can then be recorded via a powerlab and labchart data acqusition system, which is purchased separately.
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. Using cellattached patch clamp to monitor neuronal activity. Remove xenopus oocytes from their incubation solution with a polished pasteur pipette and place into the dish. Modular design series 20 recording chambers are a family of chambers designed to fill a large number of imaging and recording needs. Patch clamp electrophysiology is used to study the electrical properties of excitable cells and ion channels.
Cellattached patch clamp recordings from unfertilized oocytes of the ascidianboltenia villosa reveal an ion channel which is activated by mechanical deformation of the membrane. After patch excision insideout configuration, the current amplitude decreased very quickly traces i. Voltage clamp technique an overview sciencedirect topics. The patchserver is able to establish all known patch clamp recording configurations automatically, from single channel cell attached, insideout and outsideout to whole cell. Use of xenopus oocytes to measure ionic selectivity of pore. Cell attached patch recordings from single cx46 hemichannels in xenopus oocytes. The vitelline membrane was removed either mechanically after briefly exposing the oocyte to a hypertonic solution, or by enzyme treatment with pronase. The ideal series resistance of the patch pipette is between 11. The pressure clamp technique was used to activate single mscca currents in cell attached membrane patches on xenopus oocytes. Protease modulation of the activity of the epithelial. To excise an insideout patch from the prepared oocyte, find the clear edge of oocyte under microscope.
Procedures for treating oocytes for patch recording are. The sensitive feedback system ensures that patch pipettes stop advancing as soon as they touch cell membrane. Oocyte was treated with ionomycin and injected with egta before patching. Patch clamp assay from single neuron down to single channel, thanks to the different recording configurations whole cell, perforated patch, cell attached, outsideout, patch clamp techniques provide the highest resolution for electrophysiological recordings in ion channel screening process. Patch clamp and perfusion techniques for studying ion. This twoelectrode voltage clamping tevc amplifier features. An oocyte is produced in the ovary during female gametogenesis. Macroscopic currents from kcnq1injected oocytes were obtained in cellattached macropatches using patch pipettes with tip diameters of 710. Patch clamp technique neurosurgery oxford academic. However, stretch consistently activated enac after apical atp was scavenged with apical hexokinase plus glucose or after p 2 receptors in the patch were blocked.
In cellattached voltageclamp mode, the capacitor provides a low impedance pathway through the patch for fast events such as action potentials, resulting in the measurement of a current with an approximately 10fold larger magnitude than would be measured at steady state for the same magnitude change in membrane potential. 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. The extrapolated reversal potential indicated that the channels are chloride. Pdf patchclamp and perfusion techniques to study ion. After inhibition by pma in cell attached recordings, application of pip2 to the cytoplasmic face of excised insideout membranes restores channel activity. With a positive pressure on the pipette, move the tip slowly towards the cell while monitoring the resistance visually or using an audio monitor that generates a tone whose frequency is. A representative 5sec segments of 60sec recordings of single channel activity at positive and negativevoltages. We are using chambers with a bath for oocyte where seal is formed and patch excised, and near it a flow bath for.
Xenopus oocytes and several types of stable or immortalized mammalian cell lines. On the discrepancy between wholecell and membrane patch. Much work is done using patches in the cell attached mode, but the resting potential of the cell is not known and neither intra nor extracellular ionic concentrations can be changed easily. Patch clamp techniques for single channel and wholecell recording. Pma reduces pip2 content in oocyte membrane and calphostinc prevents the reduction. Cellattached voltageclamp and currentclamp recording. Use oocyte dissociation solution for enzymatic defolliculation of cells. Cell attached patch clamp is easy to implement and is one of the least invasive ways to monitor and manipulate the activity of a single cell. Ionic currents were studied in xenopus laevis oocytes using the patch. Feb 23, 2015 the cell attached recording mode is the first step necessary for establishing any other patch clamp configuration. Jan 23, 2005 figure 2 compares currents in response to increasing pressure steps applied to a cell attached patch pipette solution. Its unique design allows fast intracellular current clamp measurements with sharp electrodes.
An amplifier designed for twoelectrode whole cell voltage clamping of large cells xenopus oocytes and cell structures squid axons. The patch clamp technique is a laboratory technique in electrophysiology that allows the study of single or multiple ion channels in cells. The cell attached patch clamp uses a micropipette attached to the cell membrane to allow recording from a single ion channel. Using the patch clamp software, check the resistance of the pipette our goal is approximately 34 m. To directly test this idea and gain further insight into the relationship between patch and whole oocyte mechanosensitivity, we have measured the response of. Neuroscience chapter 4 sample problems flashcards quizlet. Patch clamp recordings from cell attached patches showed that serotonin, applied locally outside the patch, caused the opening ofchannels of 3 psconductance and an average lifetime of. Patch pressure clamp recording from xenopus oocytes and chok1 cells. The technique can be applied to a wide variety of cells, but is especially useful in the study of excitable cells such as neurons, cardiomyocytes, muscle fibers and pancreatic beta cells. Cells, cell attached, and excised patches when oocytes were injected with a saturating dose of insp 3, about 0. Using cell attached patch clamp techniques, we found that mechanical stretch stimulated enac in a6 distal nephron cells in only three of nine cell attached patches.
Protein kinase c inhibits romk1 channel activity via a. Fullfunction patch clamp amplifier for both whole cells and patches true current clamp is 100% stable with bandwidth of 200khz. Aug, 2004 furthermore, one might predict that the oocyte would be more mechano. This is possible due to the fact that the patchserver utilizes standard glass electrodes and that it follows stepbystep the manual patch method. Feb 15, 2000 furthermore, one might predict that the oocyte would be more mechanosensitive than the patch since membranecytoskeleton interactions are relatively undisturbed by whole cell recording. It is carried out similarly to cell attached patch clamping, but more. This causes perforation of the patch allowing small molecules and ions, but not larger compounds, to cross the patch. The xenopus oocyte cutopen vaseline gap voltageclamp. The whole cell technique involves rupturing a patch of membrane with mild suction to provide lowresistance electrical access, allowing control of transmembrane voltage. If cellattached patches were formed before store depletion, isoc was.
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. To directly test this idea and gain further insight into the relationship between patch and whole. Molecular mechanism of voltagedependent potentiation of kcnh. Polycystinl is a calciumregulated cation channel permeable. The electrode is attached to a wire that contacts the currentvoltage loop inside the amplifier. Among other advantages, these easy to obtain, mechanically and electrically stable, largesized cells enable multiple types of electrophysiological recordings. Patch clamping is a technique in which a recording pipet is used to grip a cell membrane and record the electrical potential or flow of ions through one ion channel. We are using chambers with a bath for oocyte where seal is formed and patch excised, and near it a.
Here we present direct evidence of aqp6 channel gating using patch clamp techniques. Together with our smart ephys brands heka and warner, multi channel systems can provide you with a complete patch clamp setup, from antivibration table, highquality amplifier, puller, microscope, and faraday cage to temperature control and perfusion systems. The patch clamp method allows experimental control of the membrane potential, and it can be used to characterize the voltage dependence of membrane currents. However, once a patch was formed on an activated cell, i cl,ca measured from. Cell attached patch clamp an overview sciencedirect topics. You can get the complete setup from one source and trust in the expertise of our team. In order to form the cell attached mode, a pipette tip is placed on the surface of the cell, forming a low resistance contact seal with its membrane. Patch clamp and perfusion techniques for studying ion channels. For patch clamp you will anyway use excised most probably, outsideout patch.
Biljana jovov, albert tousson, honglong ji, deborah keeton, vadim shlyonsky, pierrejean ripoll, catherine m. This is possible due to the fact that the patchserver utilizes standard glass electrodes and that it follows stepbystep the manual patch. The patch clamp amplifier is like a twoelectrode clamp, except the voltage measuring and current passing circuits are connected in the twoelectrode clamp, they are connected through the cell. A whole cell patch clamp measures currents through the entire cell membrane. It includes a current clamp and a voltage clamp, and several patch configurations whole cell, single channel, perforated patch, etc. Since the volume of perfusion chambers oocyte well is only 4 microliter, the solution change is almost instantaneous. Pma reduces open probability of the channel in cell attached patch clamp recordings. Patch clamp traditionally uses a glass pipette, with an open tip diameter of about one micrometre, and is made such that the tip forms a smooth surfaced circle, rather than a sharp point. Patch clamp electrophysiology instruments used to evaluate ion channel behavior.
We use cell attached patch clamp for bk channel activity in vascular smooth muscle cells. Patch clamp measurements on xenopus laevis oocytes. The importance of the whole cell patch clamp technique lies in the ability to study the function of the entire population of ionic channels on a cell membrane. Wholecell currents, activated by acetylcholine or by depolarizing voltage steps had properties comparable to those observed in rat muscle.
The tip of a micropipette is placed on a cell and suction is applied though the pipette to rupture the plasma membrane that. Here are a few considerations you may want to keep in mind when patching in cell attached configuration. Atp masks stretch activation of epithelial sodium channels in. Electric circuit equivalent of the voltage clamp of the cell membrane with. The procedure involves inserting a patch electrode into the cell, penetrating the plasma membrane, crossing the cytoplasm, and finally reaching the nuclear surface. Rat brain glutamate receptors activate chloride channels. As the mean open probability of active channels observed in cell attached patches on oocytes expressing enac is often of the order of 0. The membrane under the electrode is not ruptured or physically separated from the cell, thus preserving its intracellular integrity. B the whole cell configuration allows diffusional exchange between the solution in the pipette and the cytoplasm, producing a convenient way to inject substances into the interior of the cell. Cellattached patch recordings of aqp6 expressed inxenopus laevis oocytes indicated that aqp6 is a gated channel with intermediate conductance 49 picosiemens in 100 m m nacl induced by 10. Patch clamp and perfusion techniques to study ion channels. Patch clamp amplifier axopatch 200b amplifier from molecular devices. Voltage gating and permeation in agap junction hemichannel.
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