Patch clamp

The patch clamp technique is a laboratory technique in electrophysiology used to study ionic currents in individual isolated living cells, tissue sections, or patches of cell membrane. The technique is especially useful in the study of excitable cells such as neurons, cardiomyocytes, muscle fibers, and pancreatic beta cells, and can also be applied to the study of bacterial ion channels in specially prepared giant spheroplasts. The patch clamp technique is a laboratory technique in electrophysiology used to study ionic currents in individual isolated living cells, tissue sections, or patches of cell membrane. The technique is especially useful in the study of excitable cells such as neurons, cardiomyocytes, muscle fibers, and pancreatic beta cells, and can also be applied to the study of bacterial ion channels in specially prepared giant spheroplasts. Patch clamping can be performed using the voltage clamp technique. In this case, the voltage across the cell membrane is controlled by the experimenter and the resulting currents are recorded. Alternatively, the current clamp technique can be used. In this case the current passing across the membrane is controlled by the experimenter and the resulting changes in voltage are recorded, generally in the form of action potentials. 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 channel molecules for the first time, which improved understanding of the involvement of channels in fundamental cell processes such as action potentials and nerve activity. Neher and Sakmann received the Nobel Prize in Physiology or Medicine in 1991 for this work. During a patch clamp recording, a hollow glass tube known as a micropipette or patch pipette filled with an electrolyte solution and a recording electrode connected to an amplifier is brought into contact with the membrane of an isolated cell. Another electrode is placed in a bath surrounding the cell or tissue as a reference ground electrode. An electrical circuit can be formed between the recording and reference electrode with the cell of interest in between. The solution filling the patch pipette might match the ionic composition of the bath solution, as in the case of cell-attached recording, or match the cytoplasm, for whole-cell recording. The solution in the bath solution may match the physiological extracellular solution, the cytoplasm, or be entirely non-physiological, depending on the experiment to be performed. The researcher can also change the content of the bath solution (or less commonly the pipette solution) by adding ions or drugs to study the ion channels under different conditions. 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. This type of electrode is distinct from the 'sharp microelectrode' used to puncture cells in traditional intracellular recordings, in that it is sealed onto the surface of the cell membrane, rather than inserted through it. 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 gigaohms range, called a 'gigaohm seal' or 'gigaseal'. 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. Many patch clamp amplifiers do not use true voltage clamp circuitry, but instead are differential amplifiers that use the bath electrode to set the zero current (ground) level. This allows a researcher to keep the voltage constant while observing changes in current. To make these recordings, the patch pipette is compared to the ground electrode. Current is then injected into the system to maintain a constant, set voltage. However much current is needed to clamp the voltage is opposite in sign and equal in magnitude to the current through the membrane. Alternatively, the cell can be current clamped in whole-cell mode, keeping current constant while observing changes in membrane voltage.