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The patch clamp technique is a laboratory 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, cardiomyocytes, muscle fibers and pancreatic beta cells. It can also be applied to the study of bacterial ion channels in specially prepared giant spheroplasts.
The 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. This discovery made it possible to record the currents of single ion channels for the first time, which led to proving the involvement of channels in fundamental cell processes such as action potential conduction. Neher and Sakmann received the Nobel Prize in Physiology or Medicine in 1991 for this work.[1].
Inside-out patch[edit]
In the inside-out method, a patch of the membrane is attached to the micropipette, and the cytosolic surface of the membrane is exposed to the external media, or bath [2]. One advantage of this method is that the experimenter has access to the intracellular surface of the cell via the bath. This is useful when an experimenter wishes to manipulate the environment at the intracellular surface of ion channels. For example, channels that are activated by intracellular ligands can then be studied through a range of ligand concentrations.
Excised inside-out[edit]
To achieve the excised inside-out configuration, the pipette is attached to the cell membrane in cell-attached mode forming a gigaseal, and is then retracted to break off a patch of membrane. Pulling off a membrane patch often results initially in the formation of a vesicle of membrane in the pipette tip, because the ends of the patch membrane fuse together quickly after excision. The outer face must be broken open to enter into inside-out mode; this may be done by briefly taking the membrane through the bath solution/air interface, by exposure to a low calcium solution, or by momentarily making contact with a droplet of paraffin or a piece of cured Sylgard[3]. This technique is often used to investigate single channel activity, in hopes that a single channel is located on the area of the membrane within the patch.
Inside-out whole cell[edit]
To achieve the inside-out whole cell configuration, cells are first injected into a standard patch pipette. A pressure differential is then used to force the cells to drift towards the pipette opening until they form a gigaseal. This exposes a small portion of the cell membrane to the bath solution. The pressure differential is switched to zero after a gigaseal is formed. Then, by briefly exposing the pipette tip to the atmosphere, the portion of the membrane protruding from the pipette bursts, and the cell is now in inside-out conformation, with the cell membrane located inside of the pipette. The experimenter now has access to the inside of the cell via the bath, and to the outside of the cell via the pipette solution. One advantage of this method in comparison to the excised inside-out method is the ability to measure the entire cell and obtain large current sizes[4].
Perforated patch[edit]
This variation of the patch clamp method is very similar to the whole-cell configuration. The main difference lies in the fact that when the experimenter forms the gigaohm seal, he or she does not use suction to rupture the patch membrane. Instead, the electrode solution contains small amounts of an antifungal or antibiotic agent, such as amphothericin-B, nystatin, or gramicidin, which diffuses into the membrane patch and forms small perforations in the membrane, providing electrical access to the cell interior[5]. When comparing the whole-cell and perforated patch methods, one can think of the whole-cell patch as an open door, in which there is complete exchange between molecules in the pipette solution and the cytoplasm. The perforated patch can be likened to a screen door that only allows the exchange of certain molecules from the pipette solution to the cytoplasm of the cell.
Advantages of the perforated patch method are as follows: 1. The antibiotic pores allow equilibration of small monovalent ions between the patch pipette and the cytosol whilst maintaining endogenous levels of divalent ions such as Ca 2+ and signalling molecules such as cAMP. 2. The integrity of second messenger signalling cascades is retained. 3. Reduced current rundown and stable whole-cell recording lasting longer than 1 hour.[5]
Disadvantages include: 1. A higher access resistance, relative to whole-cell, due to the partial membrane occupying the tip of the electrode. This may decrease electrical access and current resolution, and increase recording noise. 2. It can take a significant amount of time for the antibiotic to perforate the membrane. (About 15 minutes for amphothericin-B, and even longer for gramicidin and mystatin.) 3. 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 antibiotic contaminating the inside of the cell.[5]
References[edit]
- ^ "The Nobel Prize in Physiology or Medicine 1991". nobelprize.org. Nobel Media AB. Retrieved November 8, 2014.
- ^ Veitinger, Sophie. "The Patch-Clamp Technique". Science Lab. Leica Microsystems. Retrieved November 10, 2014.
{{cite web}}:|archive-date=requires|archive-url=(help) - ^ Ogden, David; Stanfield, Peter. "Patch Clamp Techniques" (pdf). utdallas.edu. pp. 53–78. Retrieved November 11, 2014.
- ^ Bowlby, Mark; Merrill, Thomas; Vasilyev, Dmitry (2005). "Development of a Novel Automated Ion Channel Recording Method Using Inside-Out Whole-Cell Membranes". Journal of Biomolecular Screening. Retrieved November 10, 2014.
- ^ a b c Linley, John (2013). "11". In Gamper, Nikita (ed.). Perforated Whole-Cell Patch-Clamp Recording (PDF) (Second ed.). Humana Press. pp. 149–157. ISBN 978-1-62703-351-0. Retrieved November 10, 2014.
External links[edit]
Category:Neurophysiology Category:Physiology Category:Electrophysiology Category:Laboratory techniques