Patch clamp
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Patch clamp technique is a technique in electrophysiology that allows the study of single or multiple ion channels in cells. The technique can be applied to a wide variety of cells but is especially useful in the study of excitable cells such as neurons, cardiac cells, 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 classic patch clamp technique, the electrode used is a glass micropipette, 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.
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[edit] Basic technique
Patch clamp recording takes place with a glass micropipette that has an open tip diameter of about one micrometre. This type of electrode is distinct from the "sharp microelectrode" used to impale cells in traditional intracellular recordings. In the early year of patch clamping, the micropipette tip was heated in a microforge to produce a smooth surface that assists in forming a gigaseal with the cell membrane. However, investigators learned that this step could be skipped in many preparations. The interior of the pipette is filled with a solution to match the ionic composition of the bath solution, as in the case of cell-attached recording, or the cytoplasm for whole cell recording mode. A chlorided silver wire is placed in contact with this solution and conducts the electrical charges to the amplifier. The investigator can change the composition of this solution or add drugs to study the ion channels under different conditions. The micropipette is pressed against a cell membrane and suction is applied to assist in formation of a high resistance seal between the glass and the cell membrane (a "gigaohm seal" or "gigaseal", since the electrical resistance of that seal is in excess of a gigaohm).
Unlike traditional voltage clamp recordings, patch clamp recording uses a single electrode to record currents. 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 level. 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.
[edit] Variations
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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.
- Cell-attached patch: The electrode remains sealed to the patch of membrane. This allows for the recording of currents through single ion channels in that patch of membrane. For ligand-gated channels or channels that are activated through the action of drug molecules, the drug of choice is usually included in the pipette solution. While the resulting channel activity can be attributed to the drug used, it is not possible to then change the drug concentration. The technique is thus limited to one point in a dose response curve per patch. Usually, the dose response is accomplished through several cells and patches. However, voltage-gated channels or channels that are activated through changes in the potential across the membrane, can be clamped at different membrane potentials using the same patch. This results in graded channel activation, and a proper I-V(current-voltage) curve can be established with only one patch.
- "Inside-out" patch: After the gigaseal is formed, the electrode is quickly withdrawn from the cell, thus ripping the patch of membrane off the cell, leaving the patch of membrane attached to the electrode exposing the intracellular surface of the membrane to the external media. This is useful when an experimenter wishes to manipulate the environment affecting the inside of ion channels. For example, channels that are activated by intracellular ligands like cGMP can then be studied through a range of ligand concentrations.
- Whole-cell recording or whole-cell patch: The electrode is left in place, but more suction is applied to rupture the portion of the cell's membrane that is inside the electrode, thus providing access to the intracellular space of the cell. The advantage of whole-cell patch clamp recording over sharp microelectrode recording is that the larger opening at the tip of the patch clamp electrode provides lower resistance and thus better electrical access to the inside of the cell. A disadvantage of this technique is that the volume of the electrode is larger than the cell, so the soluble contents of the cell's interior will slowly be replaced by the contents of the electrode. This is referred to as the electrode "dialyzing" the cell's contents. Thus, any properties of the cell that depend on soluble intracellular contents will be altered. The pipette solution used usually approximates the high-potassium environment of interior of the cell. Generally speaking, there is a "grace period" at the beginning of a whole-cell recording, lasting approximately 10 minutes, when one can take measurements before the cell has been dialyzed. Whole-cell recordings involve recording currents through multiple channels at once.
*"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.
- Perforated patch: In this variation of whole-cell recording, the experimenter forms the gigaohm seal, then adds a new solution to the electrode containing small amounts of an antibiotic, such as Amphothericin-B or Gramicidin into the electrode solution to punch small perforations on the bit of membrane attached to the electrode. This has the advantage of reducing the dialysis of the cell that occurs in whole cell recordings, but also has several disadvantages. First, the access resistance is higher (access resistance being the sum of the electrode resistance and the resistance at the electrode-cell junction). This will decrease current resolution, increase recording noise, and magnify any series resistance error. Second, it can take a significant amount of time (10-30 minutes) for the antibiotic to perforate the membrane. Third, the membrane under the electrode tip is weakened by the perforations formed by the antibiotic and can rupture. If the patch ruptures, the recording is then in whole-cell mode with the addition of antibiotic inside the cell.
[edit] See also
[edit] References
- Kandel E.R., Schwartz, J.H., Jessell, T.M. (2000). Principles of Neural Science, 4th ed., pp.152-153. McGraw-Hill, New York.
- Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85-100.
