KASHMEERA N.A.ROLL NO: 37CHRIST COLLEGE, IJK

PATCH CLAMP RECORDINGPATCH CLAMP RECORDING

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

•Erwin Neher and Bert Sakmann developed the patch clamp in the late 1970s and early 1980s. They received the Nobel Prize in Physiology or Medicine in 1991 for this work.

THE PATCH-CLAMP TECHNIQUE

Erwin NeherBert Sakmann

Germany(1991 Nobel Laureates)

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BASIC 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 patchThis 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

TYPES OF PATCH CLAMP:

Cell attached

Inside Out

Whole Cell

OutsideOut

The patch-clamp technique can be applied in several configurations

‘CELL-ATTACHED’

The cell-attached recording mode is the first step necessary for establishing any other patch-clamp configuration.

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.

Slight suction applied to the upper end of the pipette results in formation of a tight seal with a resistance of 1 to 100 Gigaohm. Such a seal with a resistance in the range of gigaohms is called “giga-seal”. Formation of a giga-seal is extremely important for reduction of noise during single-channel recordings.

CELL-ATTACHED

‘WHOLE-CELL’

Whole-cell recordings 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.

The advantage of whole-cell patch clamp recording over sharp microelectrode recording 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.

A disadvantage of this technique 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. Thus, any properties of the cell that depend on soluble intracellular contents will be altered. Generally speaking, there is a period at the beginning of a whole-cell recording, lasting approximately 10 minutes, when one can take measurements before the cell has been dialyzed.

‘OUTSIDE-OUT’

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.

. 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

‘INSIDE-OUT’

By quickly withdrawing the electrode from the cell after obtaining a cell attached configuration, the patch of membrane within the tip of the electrode can be torn from the cell while maintaining a gigaohm seal with the electrode. This configuration is referred to as an inside-out patch, in which the interior aspect of the cell membrane is exposed to the bath solution and the exterior of the membrane is exposed to the internal pipette solution. The inside -out patch configuration is useful for studying the effects of manipulating the internal environment on single ion channel function.

The patch pipette with internal recording electrode (A) and reference electrode (B) are connected to the headstage(C), which is mounted ona micromanipulator (D). Isolated cells are visualized with an inverted light microscope (E). The microscope, micromanipulator,and headstage are placed on a air table (F) to isolate these components from vibrations that may interferewith gigaseal formation, and placed within a Faraday cage (G) to shield the setup from ambient electrical noise. Theacquired analog signal from the headstage is passed to an analog to digital converter (H), where the signal is digitalizedand sent to a computer (I) for data analysis. An oscilloscope (J) is used for monitoring experiments and for data display.

Experimental set up

Air table: Uses pressurized cylinders to ‘‘float’’ the table andisolate the preparation being studied from vibrations thatcan interfere with the ability to achieve high resistance pipetteemembraneseals.

Amplifier: Amplifies current or voltage being measured and interfaceswith computer for data acquisition. Also controlsmembrane voltage (voltage clamp) or current (currentclamp) depending upon experimental protocol.

Analog to digital converter: Converts the analog signal recordedby the microelectrode to digital data that is acquired bycomputer during experiments.

Computer: Uses any of a number of proprietary software packagesfor data acquisition and analysis. These suites allow theexperimenter to control membrane voltage/current, to process the input from the amplifier (filtering, capacitancecompensation, etc.), to apply stimulus protocols, and to analyzeacquired data.

Faraday cage: Shields the headstage from stray electromagneticfields that may introduce noise into electrophysiologicrecordings. Ideally composed of a copper mesh, the Faradaycage is most effective when connected to a ground source.The microscope and headstage are most commonly placedinside the Faraday cage.

Headstage: The physical connection between the preparationbeing studied and the amplifier, the headstage has inputsfor the recording microelectrode (which comes into physicalcontact with the preparation) and ground wire (usually in theexternal bath) and interfaces with the amplifier for data acquisition,control of membrane voltage/current, and stimulationof preparation when necessary. The headstage is mounted ona micromanipulator.

Inverted microscope: Used to visualize cells/preparation beingstudied. This is important in order to identify viable cellsand to assist with appropriate orientation of microelectrodeand cell to optimize high resistance seal formation.

Micromanipulator: Used to manipulate the microelectrode(mounted on the headstage) to contact the cell/preparationbeing studied. Micromanipulators may be mechanical, electricalor hydraulic.

Figure 1. Registration of the flow of current through single ion channels using the recording technique of Neher and Sakmann. A schematically shows how a glass micropipette is brought in contact with the cell, and B, using a higher magnification, a part of the cell membrane, with ion channels, in close contact with the tip of the pipette. The interior of the pipette is connected to an electronic amplifier. C shows a channel in greater magnification with its receptor facing the exterior of the cell and its ion filter. D shows the current passing through the ion channel as it opens.

PATCH CLAMP TECHNIQUE IN ISOLATED CARDIAC MYOCYTES

APPLICATIONSAPPLICATIONS

The generation of an action potential in heart musclecells depends on the opening and closing of ion-selective channels in the plasma membrane.

The patch-clamp technique enables the investigation of drug interactions with ion-channel .

The Isolated cells are ready for experiment.

Glass micro-pipette - a tip opening of about 1 μm, is placed onto the cell.

The patch-pipette is filled with either high NaCl or KCl solution and is mounted on a micro manipulator.

A chlorided silver wire connects the pipettesolution to the head stage of an electronical amplifier.

A second chlorided silver wire is inserted into the bath and serves a ground electrode.

Whole cell patch clamping is done.

This high input resistance enables the recording of small electrical currents in the range of Picosiemens (10–12 S), which are flowing through channel-forming proteins situated in the membrane patch.

The electrical current is driven by applying an electrical potential across the membrane patch, and/or by establishing an appropriated chemical gradient for the respective ion species.

To investigate the interaction of drugs with all ion channels involved in the functioning of the heart muscle cell (K+, Na+, Ca2+ and eventually Cl– channels).

Concentration-response curves of drugs which either inhibit or activate ion channels can be recorded eitheron the single channel level or by measuring the wholecell current.

Novel Ion Channels Can Be Characterizedby a Combination of Oocyte Expressionand Patch Clamping

Cloning of human disease-causing genes and sequencing ofthe human genome have identified many genes encoding channel proteins, including 67 K channel proteins.

One way of characterizing the function of these proteins is to transcribe a cloned cDNA in a cell-free system to produce the corresponding mRNA.

Injection of this mRNA into frog oocytes and patch-clamp measurements onthe newly synthesized channel protein can often reveal its function .

This experimental approach is especially useful because frog oocytes normally do not express any channel proteins, so only the channel under study is present in the membrane. In addition, because of the largesize of frog oocytes, patch-clamping studies are technicallyeasier to perform on them than on smaller cells.

Here's what the results look like.

Time is recorded along the horizontal axis, and current along the vertical. When the channel is closed, the current is 0; when it is open, it jumps up to a tiny, fixed value. There is a quantal nature to the current flow at this level, as a single channel allows a fixed number of ions to move through it per unit time, just as opening a faucet tap allows only a certain volume of water to flow. We can see that the transition state for an ion channel between open and closed is very brief; it flicks open and closed quickly