PATCH CLAMP

Presented by:Syed KashifDepartment of PharmacologyAACP

Erwin NeherBert Sakmann

Germany(1991 Nobel Laureates)

CONTENTS1.Definition 2.Principle3.Set up4.Recording5.Variations6.Applications

Definition • The patch clamp technique is a laboratory

technique in electrophysiology that allows the study of single or multiple ion channels in 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.

• It can also be applied to the study of bacterial ion channels in specially prepared giant spheroplasts.

• The patch clamp technique is a refinement of the voltage clamp.

• Erwin Neher and Bert Sakmann developed the patch clamp in the late 1970s and early 1980s. This discovery made it possible to record the currents of single ion channels for the first time, proving their involvement in fundamental cell processes such as action potential conduction. Neher and Sakmann received the Nobel Prize in Physiology or Medicine in 1991 for this work.

Principle The principle of the method is to isolate a patch of membrane

electrically from the external solution and to record current flowing into the patch

This is achieved by pressing a fire-polished glass pipette, which has been filled with a suitable electrolyte solution, against the surface of a cell and applying light suction

The patch clamp circuit

Patch of cell membrane with ion channel

FBR

_

+Amplifier

TechnicalThe high gain operational amplifier isconnected in the circuit so that the currentflowing through the ion channel is measuredas a voltage drop across the feedback resistor(FBR). The FBR has a resistance of 50 Gallowing very small currents (10-12 A)to be measured.

Set up Patch clamp recording uses a glass 

micropipette called a patch pipette as a recordingelectrode, and another electrode in the bath around the cell, as a reference ground electrode.  Depending on what the researcher is trying to measure, the diameter of the pipette tip used may vary, but it is usually in the micrometer range. This small size is used to enclose a membrane surface area or "patch" that often contains just one or a few ion channel molecules

In some experiments, the micropipette tip is heated in a microforge to produce a smooth surface that assists in forming a high resistance seal with the cell membrane. To obtain this high resistance seal, the micropipette is pressed against a cell membrane and suction is applied. A portion of the cell membrane is suctioned into the pipette, creating an omega-shaped area of membrane which, if formed properly, creates a resistance in the 10–100 gigaohmsrange, called a "giga ohm seal" or "gigaseal".[3] The high resistance of this seal makes it possible to isolate electronically the currents measured across the membrane patch with little competing noise, as well as providing some mechanical stability to the recording

Depending on the experiment, the interior of the pipette can be filled with a solution matching the ionic composition of the bath solution, as in the case of cell-attached recording, or matching the cytoplasm, for whole-cell recording. The researcher can also change the content or concentration of these solutions by adding ions or drugs to study the ion channels under different conditions.

Variations in patch clamp 1)Cell-attached or on-cell patch: The electrode is sealed to the patch of

membrane, and the cell remains intact. This allows for the recording of currents through

single ion channels in that patch of membrane, without disrupting the interior of the cell.

For ligand-gated ion channels or channels that are modulated by metabotropic receptors, the neurotransmitter or drug being studied is usually included in the pipette solution, where it can contact what had been the external surface of the membrane.

2)Whole-cell recording or whole-cell patch: Whole-cell recordings, in contrast, involve recording

currents through multiple channels at once, over the membrane of the entire cell.

The electrode is left in place on the cell, but more suction is applied to rupture the membrane patch, thus providing access to the intracellular space of the cell.

Advantage: Is that the larger opening at the tip of the patch clamp electrode provides lower resistance and thus better electrical access to the inside of the cell.

Disadvantage:Is that the volume of the electrode is larger than the cell, so the soluble contents of the cell's interior will slowly be replaced by the contents of the electrode. This is referred to as the electrode "dialyzing”the cell's contents.

3)Outside-out patch: After the whole-cell patch is formed, the electrode

can be slowly withdrawn from the cell, allowing a bulb of membrane to bleb out from the cell.

When the electrode is pulled far enough away, this bleb will detach from the cell and reform as a convex membrane on the end of the electrode (like a ball open at the electrode tip). with the original outside of the membrane facing outward from the electrode.

Single channel recordings are possible in this conformation if the bleb of membrane is small enough.

Outside-out patching gives the experimenter the opportunity to examine the properties of an ion channel when it is isolated from the cell, and exposed to different solutions on the extracellular surface of the membrane.

Advantage:The experimenter can perfuse the same patch with different solutions.

Disadvantage:It is more difficult to accomplish, as more steps are involved in the patching process.

4)Perforated patch:In this variation of whole-cell recording, the experimenter forms the gigohm seal, but does not use suction to rupture the patch membrane.

Instead, the electrode solution contains small amounts of an antifungal or antibiotic agent, such as amphothericin-B, nystatin, or gramicidin.

As the antibiotic molecules diffuse into the membrane patch, they form small perforations in the membrane, providing electrical access to the cell interior.

5)Loose patch: Loose patch clamp is different in that it employs

a loose seal rather than the tight gigaseal used in the conventional technique.

Advantages: Is that the pipette that is used can be repeatedly removed from the membrane after recording, and the membrane will remain intact.

This allows for repeated measurements in a variety of locations on the same cell without destroying the integrity of the membrane.

Disadvantage: Is that the resistance between the pipette and the membrane is greatly reduced, allowing current to leak through the seal.

Procedure Acute brain slices / cultured cells / enzymatically isolated cells

should be superfused in ACSF / extracellular solution and continuously gassed with carbogen (5% CO2/95% O2) for at least 2 h at room temperature before recording.

Pull recording microelectrodes to an input resistance of 5–8 MΩ. ▲ CRITICAL STEP

Set the bath application system to run at 1–2 ml per minute. Place the slice/cells in.

Fill the recording microelectrode with electrode solution. If documenting cellular morphology post hoc is desired, include the

intracellular dye filling of choice in the micropipette solution. Available dyes include Lucifer yellow, Cell Tracker, biocytin, Alexa biocytin, neurobiotin, etc.

Place the microelectrode in the pipette holder. Apply positive pressure using a 10-ml syringe by displacing the plunger about 1 ml. ▲ CRITICAL STEP

Set the amplifier to voltage clamp and apply a test pulse of 5–10 msec and 20 mV amplitude. Slowly approach the area of interest until there is an obvious change in the test pulse amplitude.

Once an obvious and steady change in microelectrode resistance is obtained, release the positive pressure rapidly. ▲ CRITICAL STEP

Obtain a GΩ seal spontaneously. If not, briefly apply light suction by mouth until the resistance reaches at least 1 GΩ.

Once a GΩ seal has been formed, proceed to obtain the desired patch-clamp configuration.◦ Cell-attached configuration: Upon acquiring a GΩ seal one

can proceed with the experiment. In this configuration, the microelectrode solution should resemble extracellular medium.

◦ Inside-out configuration: After obtaining a GΩ seal, slowly pull the pipette away from the cell. Eventually, a small piece of cell membrane will be detached from the cell surface without losing the GΩ seal. The microelectrode solution should resemble extracellular medium in this configuration also.

◦ Whole-cell configuration: In this configuration, the microelectrode solution should

approximate intracellular ionic composition. To record in whole-cell mode, change the voltage clamp to a

negative voltage close to the cell resting potential (–60 mV for radial glial cells) and correct for fast capacitance. Apply continuous light suction by mouth until the membrane breaks as evidenced by a change in the capacitance and the test pulse current. ▲ CRITICAL STEP

If doing perforated patch recordings, front-fill the microelectrode with electrode solution without antibiotic, back-fill with electrode solution containing antibiotic. After a GΩ seal is obtained, simply set the voltage clamp near the resting potential and wait for the resistance to slowly decrease and stabilize.

◦ Outside-out configuration: After obtaining a whole-cell recording, very slowly withdraw the pipette until resistance increases greatly, indicating formation of an excised membrane ‘bleb.’

Analyze recordings. Most acquisition software comes bundled with analysis software.

Applications For the evaluation of antiarrhythmics agents.

In kidney cells.

Used for isolated ventricular myocytes from Guinea pigs to study a

cardio selective inhibition of the ATP sensitive potassium channel.

To identify multiple types of calcium channels.

To measure the effect of potassium channel openers.

Used in the molecular biology.

Voltage clamp studies on sodium channels.

Used to investigate a wide range of electrophysiological cell

properties.

Measurement of cell membrane conductance.

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