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Patch clamp technique is a technique in electrophysiology that allows the study of individual ion channels in cells. The technique is used to study excitable cells such as neurons, muscle fibers and the beta cells of the pancreas. It can also be applied to the study of bacterial ion channels in specially prepared giant spheroplasts. In classical patch clamp technique, the electrode used is a glass pipette, but planar patch clamp uses a flat surface punctured with tiny holes.
Patch clamp technique is a refinement of the voltage clamp. 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.
Patch clamp traditionally uses a glass pipette, with an open tip diameter of about one micrometre, and is made such that the tip forms a smooth surfaced circle, rather than a sharp point. This style of electrode is known as a "patch clamp electrode" and is distinct from the "sharp microelectrode" used to impale cells in traditional intracellular recordings. The interior of the pipette is filled with different solutions (usually called the pipette solution) depending on the specific technique or variation used (see following). For example, with whole cell recordings, a solution that approximates the intracellular fluid is used. A metal electrode in contact with this solution conducts the electrical changes to a voltage clamp amplifier. The researcher can change the composition of this solution or add drugs to study the ion channels under different conditions. The patch clamp electrode is pressed against a cell membrane and suction is applied to the inside of the electrode to pull the cell's membrane inside the tip of the electrode. The suction causes the cell to form a tight seal with the electrode (a so-called "gigaohm seal", since the electrical resistance of that seal is in excess of a gigaohm).
Unlike traditional voltage clamp recordings, the patch clamp recording uses a single electrode to voltage clamp a cell. This allows a researcher to keep the voltage constant while observing changes in current. Alternately, the cell can be current clamped, keeping current constant while observing changes in membrane voltage.
Several variations of the basic technique can be applied, depending on what the researcher wants to study. The inside-out and outside-out techniques are called "excised patch" techniques, because the patch is excised (removed) from the main body of the cell. Cell-attached and both excised patch techniques are used to study the behavior of ion channels on the section of membrane attached to the electrode, while whole-cell patch and perforated patch allow the researcher to study the electrical behavior of the entire cell.
*"Outside-out" patch: After the aforementioned 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 ball of membrane on the end of the electrode, with the outside of the membrane being the surface of the ball. Outside-out patching gives the experimenter the opportunity to examine the properties of an ion channel when it is protected from the outside environment, but not in contact with its usual environment. In this conformation, the experimenter can perfuse the same patch with different solutions, and if the channel is activated from the extracellular face, a dose-response curve can then be studied. Single channel recordings are possible in this conformation if the bleb of membrane is small enough. This is the distinct advantage the outside-out patch variation possesses relative to the cell-attached method. However, it is more difficult to accomplish as more steps are involved in the patching process.
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Patch_clamp". A list of authors is available in Wikipedia.|