Voltage V.S Current Clamp
I saved some of his equipment from being trashed (in Woods Hole). Patch clamping is voltage clamp but cleverly adapted so that you only need .. on the hERG channel waveform or the current–voltage relationship when compared to PSS. The patch clamp technique is a laboratory technique in electrophysiology used to study ionic Patch clamping can be performed using the voltage clamp technique. In this case the current passing across the membrane is controlled by the . on the outside of the patch of membrane, in relation to the patch electrode. The Patchliner is an automated patch clamp system capable of recording up to 8 have been used in the voltage clamp and current clamp modes with the view to approximately 40% and gave an IC50 of μM calculated from the graph.
Move the plate or flask gently to detach all cells from the bottom do not hit the flask. Carefully resuspend the cells by gentle pipetting two times max. Harvesting protocol for human iPS cell-derived iCell cardiomyocytes. Amounts are listed for a T25 flask exemplarily: Despite removing the liquid, there is still enough Trypsin on the surface to detach the cells. Check the detachment of cells under a microscope after 3 min. Move the plate or flask gently to detach all cells from the bottom do not hit the flask, tap gently if required.
If cells are not detached return the flask to the incubator. External solution except for recordings shown in Figure 9 B: The graphs are shown on the same scale. Current traces of TRPV3 activated by heated solution. External solution was heated to the temperature shown and applied to the cell. A Representative traces of a Cor. At cardiomyocyte recorded in the voltage clamp mode and block by increasing concentrations of TTX.
B Corresponding concentration response curve with a calculated IC50 of 1. C Corresponding time plot of the experiment showing the stability of the recording. Current amplitude was reduced step by step upon application of the increasing concentrations. A washout of TTX was performed at the end of experiment.
D Representative traces of highly reproducible APs as recorded in the current clamp mode. Sweep interval between each stimulus was 10 s. Traces were recorded over 15 min, the first and the last 4 APs are shown. The same experiment as shown in A was performed on the semi-automated patch clamp system Port-a-Patch E. The corresponding IC50 was calculated to be 1. G Comparison of an AP recorded with the Port-a-Patch top trace and using a conventional patch clamp rig lower trace. Gray line indicates 0 mV.
B Corresponding concentration response curve for an average of four cells reveals an IC50 of 2. C In the current clamp mode, a TTX concentration-dependent reduction of the amplitude as well as a concentration-dependent AP shortening was observed.
The Patch-Clamp Technique
The IC50 was calculated to be 5. Whole cell patch clamp recordings were conducted as previously described Farre et al. Currents were elicited using voltage protocols in the voltage clamp mode.
For heat activation of TRPV3 channels, external solution was heated to the temperature indicated and applied to the cell. Current amplitude at 90 mV was used for analysis. APs were generated using a depolarizing pulse to the threshold at which an AP was elicited.
The AP traces shown represent an average response of four recorded APs. The APs were normalized to the time point of the beginning of the upstroke. Patch clamp amplifier for the Patchliner: Therefore, it is a desirable option to be able to study ion channels at elevated temperature.
To meet this need, several heating elements were introduced into the Patchliner. The surrounding of the planar patch clamp chip can be heated to maintain constant, physiological temperature. The solution, which is pipetted onto the cell, can also be heated separately. Heating of the solution, which is applied to the cell takes One compound, which has been shown to have an increase in potency at physiological temperature, is erythromycin.
Erythromycin is a macrolide antibiotic, which can cause QT prolongation and cardiac arrhythmia. However, at RT erythromycin is much less potent. This is in good agreement with the literature Guo et al.
Nanion Technologies - Products - Port-a-Patch
Typically, single concentrations of erythromycin were applied to each cell. This is in excellent agreement with values reported in the literature Stanat et al. Heat activation of TRPV3 Transient receptor potential TRP channels are an important class of receptors found widely distributed throughout the mammalian central and peripheral nervous systems.
They have been shown to be activated by many stimuli including temperature, mechano-stimulation, divalent cations, and pH for review see Clapham, TRP channels are receiving much attention as potential targets for the treatment of, for example, chronic pain, asthma, and diabetes insipidus Clapham, ; Gudermann and Flockerzi, This is in good agreement with values reported in the literature Chung et al.
TRPV3 receptors can also be activated by temperature. Figure 4 shows the activation of TRPV3 channels by increasing temperature.
Parallel Current Clamp Measurements The Patchliner was used in combination with stem cell-derived cardiomyocytes, a standardized and pure cardiac myocyte model. This is a new opportunity because cardiac toxicity can now be evaluated in a cell type which better represents the real physiological environment compared with a cell line overexpressing a particular ion channel. Patch clamp measurements with cardiac myocytes are sophisticated experiments that were, until now, only possible using conventional patch clamp using freshly isolated cardiac myocytes from animals.
Stem cell-derived cardiac myocytes are commercially available, a pure population and ready-to-use resulting in a quicker cell preparation time and better reproducibility of the assay.
The Patchliner was used for pharmacological experiments in both voltage clamp and current clamp modes so that the effects of cardiotoxic compounds could be directly investigated on both ion currents and APs.
The eight-channel system Patchliner and the one-channel system Port-a-Patch Farre et al. We used mouse stem cell-derived Cor. At cardiomyocytes and human iPS cell-derived iCell cardiomyocytes.
The Patch-Clamp Technique: Leica Microsystems
Cole discovered that it was possible to use two electrodes and a feedback circuit to keep the cell's membrane potential at a level set by the experimenter. Cole developed the voltage clamp technique before the era of microelectrodesso his two electrodes consisted of fine wires twisted around an insulating rod. Because this type of electrode could be inserted into only the largest cells, early electrophysiological experiments were conducted almost exclusively on squid axons.
A personal photo of Kenneth Cole, given to Dr. Walter Woodbury Squids squirt jets of water when they need to move quickly, as when escaping a predator. The squid giant axon was the first preparation that could be used to voltage clamp a transmembrane current, and it was the basis of Hodgkin and Huxley's pioneering experiments on the properties of the action potential. The use of voltage clamps in their experiments to study and model the action potential in detail has laid the foundation for electrophysiology ; for which they shared the Nobel Prize in Physiology or Medicine.
Transmembrane voltage is recorded through a "voltage electrode", relative to groundand a "current electrode" passes current into the cell. The experimenter sets a "holding voltage", or "command potential", and the voltage clamp uses negative feedback to maintain the cell at this voltage.
The electrodes are connected to an amplifier, which measures membrane potential and feeds the signal into a feedback amplifier. This amplifier also gets an input from the signal generator that determines the command potential, and it subtracts the membrane potential from the command potential Vcommand — Vmmagnifies any difference, and sends an output to the current electrode.
Whenever the cell deviates from the holding voltage, the operational amplifier generates an "error signal", that is the difference between the command potential and the actual voltage of the cell. The feedback circuit passes current into the cell to reduce the error signal to zero.
Thus, the clamp circuit produces a current equal and opposite to the ionic current. Variations of the voltage clamp technique[ edit ] Two-electrode voltage clamp using microelectrodes[ edit ] Two-electrode voltage clamp The two-electrode voltage clamp TEVC technique is used to study properties of membrane proteins, especially ion channels.
The large size of these oocytes allows for easy handling and manipulability. The microelectrodes are filled with conductive solution and inserted into the cell to artificially control membrane potential. The membrane acts as a dielectric as well as a resistorwhile the fluids on either side of the membrane function as capacitors.
Current readings can be used to analyze the electrical response of the cell to different applications. This technique is favored over single-microelectrode clamp or other voltage clamp techniques when conditions call for resolving large currents.
The high current-passing capacity of the two-electrode clamp makes it possible to clamp large currents that are impossible to control with single-electrode patch techniques. However, TEVC is limited in use with regard to cell size.
It is effective in larger-diameter oocytes, but more difficult to use with small cells.
Additionally, TEVC method is limited in that the transmitter of current must be contained in the pipette. It is not possible to manipulate the intracellular fluid while clamping, which is possible using patch clamp techniques. Cole's voltage clamp used a long wire that clamped the squid axon uniformly along its entire length.
TEVC microelectrodes can provide only a spatial point source of current that may not uniformly affect all parts of an irregularly shaped cell. Dual-cell voltage clamp[ edit ] The dual-cell voltage clamp technique is a specialized variation of the two electrode voltage clamp, and is only used in the study of gap junction channels.
When two cells in which gap junction proteins are expressed either endogenously or via injection of mRNAa junction channel will form between the cells. Since two cells are present in the system, two sets of electrodes are used. A recording electrode and a current injecting electrode are inserted into each cell, and each cell is clamped individually each set of electrodes is attached to a separate apparatus, and integration of data is performed by computer.
To record junctional conductancethe current is varied in the first cell while the recording electrode in the second cell records any changes in Vm for the second cell only. The process can be reversed with the stimulus occurring in the second cell and recording occurring in the first cell. Since no variation in current is being induced by the electrode in the recorded cell, any change in voltage must be induced by current crossing into the recorded cell, through the gap junction channels, from the cell in which the current was varied.
Continuous single-electrode clamp SEVC-c technique is often used with patch-clamp recording. Discontinuous single-electrode voltage-clamp SEVC-d technique is used with penetrating intracellular recording. In whole-cell measurements the researcher can choose between two configurations: Moreover, it is also possible to record currents only from a small patch instead of the whole cell. This raises the chances of recording single channels.
The patch can be orientated in two different directions inside the patch pipette. To achieve the inside-out configuration the patch pipette is attached to the cell membrane and is then retracted to break off a patch of membrane Figure 3. In this case the cytosolic surface of the membrane is exposed.
This is often used to investigate single channel activity with the advantage that the medium that is exposed to the intracellular surface can be modified. If the aim is to study the influence of extracellular cues such as neurotransmitters, the outside-out configuration Figure 3 should be chosen. In this case the pipette is retracted during the whole-cell configuration, causing a rupture and rearrangement of the membrane.
In this configuration the extracellular surface is exposed and thus extracellular cues can easily be applied. The four recording methods for patch-clamp: When the pipette is in closest proximity to the cell membrane, mild suction is applied to gain a tight seal between the pipette and the membrane. By applying another brief but strong suction, the cell membrane is ruptured and the pipette gains access to the cytoplasm. In the cell-attached mode, the pipette is retracted and the patch is separated from the rest of the membrane and exposed to air.
The cytosolic surface of the membrane is exposed. In the whole-cell mode, the pipette is retracted resulting in two small pieces of membrane that reconnect and form a small vesicular structure with the cytosolic side facing the pipette solution. Patch me if you can — What is the patch-Clamp Technique? The ion channel of interest can also be isolated and expressed heterogeneously in a common cell line e.
Depending on the sample, either an inverted cultured cells or an upright fixed stage microscope for slices with a stable platform is needed. If cells in acute slices are investigated, an infrared DIC is recommendable to visualize the membrane.
The microscope should be placed on an anti-vibration table because any movement could be fatal to the seal between the pipette and the membrane.
A micromanipulator is needed to move the pipette precisely. Very fine pipettes are formed by heating and pulling small glass or quartz capillary tubes. The tip of the pipette is heat-polished in a microforge to gain a high-resistance seal onto the membrane.
The pipette is filled with a solution that resembles either the extracellular solution or the cytoplasm, depending on the recording mode. The pipette is mounted on a micromanipulator to permit precise movements towards the cell membrane. For conductance of the current a chlorided silver wire is used.