Patch-clamp technique is the name given to an electrophysiological measurement technique. It allows ionic currents to be measured through individual channels within a plasma membrane.
What is the patch-clamp technique?
The patch clamp technique or patch clamp method belongs to electrophysiology, which is a branch of neurophysiology dealing with the electrochemical transmission of signals in the nervous system. With the aid of this method, it is possible to visualize individual ion channels in the cell membrane of a body cell. This involves the measurement of currents of a few picoamperes. The patch-clamp technique was first described in 1976 by the German biophysicist Erwin Neher and the German physician Bert Sakmann. The two scientists were awarded the Nobel Prize in Physiology or Medicine in 1991 for the development of the clamp-patch technique. Thus, electrophysiological research was virtually revolutionized by the patch-clamp technique because it opened up the possibility of observing electrical behavior at the membrane proteins of individual molecules. The term patch comes from the English language and means “patch”. It refers to a small membrane section underneath the patch pipette, which is used as a measuring electrode. During the measurement process, the membrane patch is fixed or clamped (to clamp) to specified potentials.
Function, effect and objectives
The patch-clamp technique is an electrophysiological analysis method. It is based on the biological fact that cells have a large number of pores and ion channels. Different ion concentrations or charges occur inside and outside of each cell, which depends on the physiological state of the cell. The lipid bilayer of the membrane is not permeable to water molecules as well as ions. Nevertheless, an exchange of charged particles takes place across the cell membrane at irregular intervals. The reason for this is the voltage dependence of the ion channels. If a certain membrane potential is reached, the channels are opened according to the principle of “all or nothing”. This is exactly where the patch clamp technique comes in. In this way, a measuring pipette is advanced to an ion channel without penetrating the cell membrane. In this way, the local electrical potential can be determined precisely. Leakage currents, which could affect the result of the measurement, can usually be avoided by electrically extremely tight connections between the pipette edge and the cell membrane. The patch clamp method is based on the voltage clamp technique. This technique was developed in the 1930s by the American biophysicist Kenneth Stewart Cole (1900-1984) to measure currents on nerve cells that are intact. In the voltage clamp, the insertion of two electrodes into a cell takes place to provide a command or holding voltage. At the same time, another electrode is used to record the currents that occur across the membrane. If neurophysiologists want to know about the flow of electrical currents through specific areas of a nerve cell membrane, they use the patch clamp technique. To do this, they use a fine glass pipette that is placed on the outside of the cell. Negative pressure can be created by aspirating it with the help of a hypodermic syringe. This procedure causes the membrane to bulge slightly at the corresponding location. The negative pressure ensures that the glass is attached to the membrane. This results in electrical isolation of the small membrane spot in the pipette from the rest of the membrane. To measure the electrical currents, the neurophysiologists use a patch clamp amplifier. This is a special measuring device. In the ideal case, the scientist can use the device to obtain information about the electrical properties of the individual ion channels. The ion channels regulate, for example, the inflow and outflow of sodium ions, which are positively charged, in the nerve cells. The investigation takes place on the cells of humans, plants or animals. The patch-clamp method is usually performed at a measuring station that includes various devices. On the vibration-damped measuring table there is a so-called Faraday cage, which serves as an electrical shield. Furthermore, an optical microscope including a micromanipulator is available to bring the patch pipette into position.In addition, the pipette holder has a connection to a preamplifier, while the sample holder is connected to a bath electrode. The patch clamp amplifier functions to amplify the preamplifier signal. A monitor is also provided to observe the DUT as well as the patch pipette. In most cases, a computer and several data storage devices are also available at the measuring table to enable digital recording.
Risks, side effects, and hazards
There are no risks associated with the patch-clamp technique. For example, cells from humans, animals, or plants are not examined until after they have been removed. Unrestricted access to the outer cell membrane rarely exists. For this reason, it is often necessary to prepare the cells for the patch-clamp method. After filling the patch pipette, it is clamped in a micromanipulator. This is connected to the patch clamp amplifier and gently pressed onto a cell that is intact. The process can be followed with a monitor or microscope. Underneath the pipette sits a piece of membrane called a membrane patch. The slight negative pressure created at the back end of the pipette provides a strong connection between the pipette and the membrane. This process results in the creation of an electrical resistance between the external solution and the pipette interior of several gigaohms. Scientists also refer to this as “gigaseal,” which allows the cell-attached configuration of the patch-clamp method to be achieved. The current flowing through an ion channel in the patch also flows through the contents of the pipette due to the high gigaseal resistance. An electrode connected to the amplifier is immersed in the solution of the pipette, allowing the activities of individual ion channels within the patch membrane to be measured.
