Patch clamp

The patch -clamp technique (english patch clamp technique) is a measurement method in electrophysiology, with which the current can be represented by single ion channels in the cell membrane of a cell, thereby measured currents of a few picoamperes (10-12 amps). This technique was first described in 1976 by Erwin Neher and Bert Sakmann. For her work on the function of individual cellular ion channels, which they had carried out with this technique, they were awarded the 1991 Nobel Prize in Physiology or Medicine. By the possibility to monitor the electrical behavior of the membrane proteins in a single molecule, has revolutionized the patch clamp technique, the electro- physiological research.

The term patch refers to the small membrane neck (English patch: patch ) under the patch pipette, which serves as the measuring electrode at the same time. During the measurement of the membrane patch is held at a predetermined potential ( engl. to clamp: fasten clamp ). Depending on whether after putting the patch pipette this membrane region extracted from the cell or is measured at the intact cell, certain configurations are distinguished in the patch -clamp technique.

Overview

The patch -clamp technique is based on the technique of voltage clamp on (english voltage clamp; engl to clamp. Attach, clamp; engl voltage: voltage. ), Which in the 1930s by Kenneth Cole and HJ Curtis for measuring currents intact nerve cells has been developed. In this method, two electrodes are engraved into the cell: one is used to specify a holder or command voltage, while the occurring currents are recorded across the membrane to a further electrode. By means of this technique, the sum of all the individual currents through the cell membrane is measured, the resolution of individual portions is not possible.

All of the cells included in the plasma membrane surrounding called ion channels - proteins which may act such as pores for the ions of dissolved salts. Ion channels are found also in the membranes inside the cell, in plants, for example in the tonoplast - the membrane that separates the Zentralvakuole from the cytoplasm. In living cells, these ions are present on the outside and inside of the cell membrane in unequal concentration; This uneven distribution leads to a voltage across the membrane, the transmembrane potential. Ion channels play an important role for the maintenance of the resting membrane potential of the cell and for possible changes that, if they pass by leaps and bounds, action potentials are called. Action potentials serve the conduction of electrical stimuli in the organism (for example, the excitation line ). The resting membrane potential typically assumes values ​​of about -70 mV for animal and up to 200mV in plant cells.

As the carrier substance of biological membranes, the lipid bilayer, of water and dissolved ions is substantially impermeable, has been suggested earlier, that a change in conductivity can only be achieved by opening or closing of specific membrane proteins, ion channels. The two British scientists Alan Hodgkin and Andrew Huxley developed a model that the presence of voltage-dependent gates (English gates ), which open after the all-or- nothing principle completely or close, predicted in the cell membrane. 1969 was the first time evidence for the bacterial membrane protein gramicidin A, this gate function.

With voltage-clamp measurements, however, it was not possible to study the molecular mechanisms of these gates, the ion channels. The method has been developed significantly in the patch -clamp technique. Here, the electrode is not inserted into the cell, but is mounted directly on the cell membrane. Through the very small outlet opening of the pipette ( about 1 micron ) is achieved with high probability of detecting a single ion channel protein. Simultaneously, a electrically tight bond between the glass edge of the measuring electrode and the membrane is made such that leakage current is negligible. Thus, very small currents to be measured ( in the order of 5 Pa). The electrode is used at the same time for presetting the holding or command voltages as well as to measure the ion current. This dual task is possible through the use of a switched virtual ground operational amplifier in the measurement electronics; a second electrode having cell contact is not required.

Overall, the patch -clamp technique high demands on the equipment used and the measurement electronics ( low noise, long term stability ), at the same time it requires the experimenter pronounced skill with equipment and biological material.

Equipment and Materials

Measuring station

Usually a measuring station is set up for working with the patch -clamp technique, which is typically equipped with various equipment. On a vibration-damped measuring table is a Faraday cage for electrical shielding. Inside is an inverted microscope with a micromanipulator to position the patch pipette. The pipette holder is connected to the preamplifier, the sample holder with the bath electrode. The signal of the pre-amplifier is amplified in the patch-clamp amplifier. A monitor is used for monitoring of the measurement object and the patch pipette through the microscope camera. Most have a computer and data storage is for evaluating and recording the electrical signals directly at the measuring station for digital recording. The behavior of the cell may also be recorded with a video recorder.

Measuring electrode ( patch pipette )

The measuring electrode is made ​​of a glass capillary, which is drawn out to very thin when heated. The glass material usually used borosilicate glass; the exhaustion can be automated with appropriate equipment (English puller ); thereby to parameters such as heating temperature and adjust tension. Often the tip of the pipette is still heat polished after drawing, making very smooth edges are generated at the top, which favor the formation of an electrically tight connection ( gigaseal ) between the pipette and the membrane.

For the electrode, the pipette by filling it with a conductive solution in which plunges silver wire coated with silver chloride. The filled patch pipette is tensioned in the holder and connected to the preamplifier mounted there, together with a further silver wire electrode located in the bath solution. The short distance between the measuring electrode and the amplifier is necessary to reduce the overlap of the very small currents measured by external noise to a minimum. The resistance of the measuring electrode is filled with the solution is typically 1 to 5 milliohms. An additional coating the patch pipette with silicone elastomer reduces the noise of the measuring system and prevents its electric capacity increased by wetting with the bath solution. The tip remains free to about 50 microns.

A gigaseal normally develops only when fresh (a few hours old) patch pipettes prepared for the measurement are used.

Preparing the cells

The outer cell membrane of cells is seldom fully accessible; for patch-clamp measurements, the cells therefore must often be prepared first.

Animal cells

Animal cells can be enzymatically detached from the basal lamina and freed from residues of the connective tissue. This step can sometimes omitted when cells are grown in cell culture.

Plant cells

Plant cells are surrounded by a cell wall, it is usually removed by enzymatic digestion with cellulase and hemicellulase. Once the components of the cell wall is sufficiently reduced, the cells can be removed by simple, gentle agitation or by conversion into a hypoosmotic solution of the remaining residues. Such protoplasts ( cells without cell wall ) often begin immediately after removal from the enzyme bath again with the formation of new cell wall material, the measurement should be carried out as quickly as possible after the Protoplastieren.

MEASUREMENT

The filled patch pipette is clamped in a micromanipulator connected to the patch-clamp amplifier and then pressed under visual control (observation in the microscope or on a monitor connected to it ) carefully on an intact cell. Below the pipette within the diameter of the tip, there is a piece of membrane - the patch or membrane patch (English patch - patch ). A strong connection ( seal ) is then removed by gentle vacuum is applied to the rear end of the pipette is generated between the membrane and the pipette. This results in an electrical resistance of the order of several giga ohms (109 ohms), the so-called " gigaseal " between the interior of the pipette and the external solution (English to seal - seal ). With the production of gigaseals the so-called cell-attached configuration of the patch -clamp technique is reached ( engl. to attach - attach, fasten on, fasten on ). Due to the high resistance of the gigaseal has a current flowing through ion channels in the patch, also flow through the contents of the pipette. In the pipet solution immersed an electrode that is connected to a sensitive amplifier. This makes it possible to measure the activity of a single ion channel in the membrane of the patch. Both the cell membrane, which includes the patch, and the interior of the cell will remain intact in this configuration.

By further application of a vacuum at the end of the pipette or short pulses of electrical power to the electrode in the patch pipette can be opened while the gigaseal intact. Now exists between the interior of the pipette, and the interior of the cell continuity while both are insulated from the outside solution through the high resistance of the gigaseal. This configuration of the patch clamp technique is referred to as whole-cell configuration (English whole cell - whole cell). In this configuration, is derived from the total cell membrane. Since the pipette solution fills the interior of the cell, it needs to be similar in composition to the cytosol. At the same time, this configuration offers the opportunity to manipulate the cell from the inside of the pipette solution.

If is not opened after reaching the cell-attached patch configuration, but the pipette is gently withdrawn from the cell, is located under the tip of the pipette part of the membrane of the cell dissolves and remains in the pipette. In this case, now, the former inner side of this membrane piece outwardly in the bath solution, while the outer side of the former membrane piece is located inside the pipette. This is the so-called inside-out configuration. Similar to the cell-attached configuration, it allows the measurement of specific ion channels in the membrane of the pipette tip piece. In contrast to this, however, the medium can be manipulated to the inside of the membrane in the inside-out configuration. If you fill the pipette with a solution simulating the extracellular milieu, one can study the behavior of ion channels as a function of the composition of the cytosol. In addition, there is the possibility of the investigation of single ion channels using the outside-out configuration. In this case, the bulging out of the patch pipette opening and the outer side of the membrane is now located in the bath solution. In this case the environment of the extracellular space can be modulated, whereas the cytosolic environment is maintained.

It is even possible with this method to measure the function of cells in their natural environment in a living organism (in vivo).

Planar patch clamp

Planar patch clamp is a new method that was developed to increase the throughput in electrophysiology, to meet, among other things due to the growing need for patch clamp measurements in pharmaceutical research.

Instead of positioning a pipette on an adherent cell, the cell suspension is pipetted onto a chip, in which a microstructured aperture was placed.

A single cell is then drawn by negative pressure at the hole, and an electrically tight connection between the cell and formed glass ( gigaseal ). The planar geometry features compared with the conventional patch-clamp method, a number of advantages:

• The integration of microfluidic channels allows automated drug addition, as is required in pharmaceutical research.
• The system is accessible for optical and scanning probe microscopy methods.
• Perfusion of Intrazellulärseite is much easier possible whereby active ingredients can be better examined, act intracellularly.
• For the measurements, no special training is required.
• Non-adherent cells (such as red blood cells), which classically can be studied very hard, are planar much easier to patch.
• The measurement setups can be miniaturized:

While a classical structure can easily fill half a laboratory room, can be a planar structure realized as a tabletop unit.

The advantages are some disadvantages:

• Cells such as macrophages or neurons adhere firmly to the surface on which they were cultured. In the enzymatic separation must be ensured that the proteases do not affect the channel protein on the cell surface. So Adherent cells can not be patched under strictly physiological conditions. The most extensive deployment of Planar patch systems therefore in transfected cell cultures.
• Since the cells have to be separated, no measurements on tissue sections are possible.
• It must be ensured that the cell suspension is homogeneous, because the patched cell is "blind" chosen from the ensemble. Thus, the cell can not be selected on the basis of their shape or a fluorescent label.
• Despite the high degree of miniaturization, the cost per measuring channel are in a similar range as in the conventional patch clamp. Highly-Parallel systems therefore are only viable at correspondingly high passage numbers.