The Patch-Clamp Technique: Important Tool for The Investigation of Cell Behavior
Especially in neuroscience, the physiology of ion channels has always been a major topic of concern. The development of patch clamp technology in the late 1970s brought new prospects to electrophysiologists. It can record not only the high-resolution current of the entire cell, but also the high-resolution current of the excised cell mass, and can even investigate single-channel opening events. However, electrophysiology is still one of the most challenging methods in daily laboratory work due to its complex technical, physical and biological background, the need for highly sensitive equipment, and the large amount of skills required by experimenters.
Electrophysiology is a technology pioneered in a specialized biophysics laboratory, which has now been extended to basic biology and medical research, and has become one of the most important tools for studying the behavior of single cells or entire cell networks in the nervous system.
Patch-clamp technology allows the study of small-scale or even single ion channels. Therefore, it has particular potential in the study of excitatory cells such as neurons, cardiomyocytes and muscle fibers. An ion channel can conduct about 10 million ions per second. However, there are currently only a few picoamperes. Recording current in this order of magnitude is very challenging not only for researchers but also for equipment. In principle, a thin glass or quartz pipette with a blunt end is sealed on the membrane.
Recording current in this order of magnitude is very challenging not only for researchers but also for equipment. In principle, a thin glass or quartz pipette with a blunt end is sealed on the membrane. The application of suction helps to develop gigaohm-level high-resistance seals. This tight seal electrically isolates the diaphragm, which means that all ions that make the flux of the diaphragm flow into the pipette, which is recorded by a silver chloride electrode connected to a high-sensitivity electronic amplifier, and the bath electrode is used to set a zero level. To prevent the membrane potential from changing, the amplifier generates a compensation current similar to the current flowing through the membrane as a negative feedback mechanism.
Measure the membrane potential of the cell and compare it with the command potential. If there is a difference between the commanded potential and the measured value, current will be injected. This compensation current will be recorded and conclusions can be drawn about the membrane conductance. The membrane potential can be manipulated independently of the ion current, which can study the current-voltage relationship of the membrane channel.
Patch-clamp experiments can be performed on cultured cells, acutely dissociated cells, or acute glass fiber sections, which can study the electrophysiological properties of the cells in their natural environment. Target ion channels can also be isolated and expressed heterogeneously in common cell lines (eg HEK293, CHO, LNCaP). Depending on the sample, it is necessary to use an inverted (cultured cell) with a stable platform or an upright fixed platform microscope (for sectioning). If examining cells in an acute section, it is recommended to use infrared DIC visualization membrane. The microscope should be placed on a shock-proof table, because any movement may cause fatal damage to the seal between the pipette and the membrane.
Patch clamp experiments are not only used in neuroscience, but also for solving various physiological problems. Over the past two decades, patch-clamp recording has become increasingly important for studying ion channels in non-excitable cells. This is also a very important method in medical research, because many diseases are related to the determination of the failure of ion channels. In pharmacological research, automatic patch clamps are used to screen effective substances for ion channel modification.