Patch clamp - Wikipedia, the free encyclopedia. A patch clamp recording of current reveals transitions between two conductance states of a single ion channel: closed (at top) and open (at bottom). 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 pancreaticbeta 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 1. 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 1. 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. 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 1. The researcher can also change the content or concentration of these solutions by adding ions or drugs to study the ion channels under different conditions. Recording. The pipette in the photograph has been marked with a slight blue color. 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. The inside- out and outside- out techniques are called . Patch Clamping: An Introductory Guide to Patch Clamp. Patch clamping is a widely applied electrophysiological technique for the study of ion. Cell- attached and both excised patch techniques are used to study the behavior of individual ion channels in the section of membrane attached to the electrode. Whole- cell patch and perforated patch allow the researcher to study the electrical behavior of the entire cell, instead of single channel currents. Read Book PDF Online Here http:// Patch Clamping: An Introductory Guide to Patch Clamp Electrophysiology Now.The whole- cell patch, which enables low- resistance electrical access to the inside of a cell, has now largely replaced high- resistance microelectrode recording techniques to record currents across the entire cell membrane. Cell- attached patch. This allows the recording of currents through single, or a few, ion channels contained in the patch of membrane captured by the pipette. By only attaching to the exterior of the cell membrane, there is very little disturbance of the cell structure. Using this method it is also relatively easy to obtain the right configuration, and once obtained it is fairly stable.
PDF File: Patch Clamping An Introductory Guide To Patch Clamp Electrophysiology - PCAIGTPCEPDF-WWRG35-9 2/4 Patch Clamping An Introductory Guide To Patch. PDF File: Patch Clamping An Introductory Guide To Patch Clamp Electrophysiology - PCAIGTPCEPDF-MAOM35-9 2/4 Patch Clamping An Introductory Guide To Patch. The resulting channel activity can be attributed to the drug being used, although it is usually not possible to then change the drug concentration inside the pipette. The technique is thus limited to one point in a dose response curve per patch. Therefore, the dose response is accomplished using several cells and patches. However, voltage- gated ion channels can be clamped successively at different membrane potentials in a single patch. This results in channel activation as a function of voltage, and a complete I- V (current- voltage) curve can be established in only one patch. Another potential drawback of this technique is that, just as the intracellular pathways of the cell are not disturbed, they cannot be directly modified either. This is useful when an experimenter wishes to manipulate the environment at the intracellular surface of single ion channels. For example, channels that are activated by intracellular ligands can then be studied through a range of ligand concentrations. To achieve the inside- out configuration, the pipette is attached to the cell membrane as in the cell- attached mode, forming a gigaseal, and is then retracted to break off a patch of membrane from the rest of the cell. 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 of the vesicle must then 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 Ca. The electrode is left in place on the cell, as in cell- attached recordings, but more suction is applied to rupture the membrane patch, thus providing access from the interior of the pipette to the intracellular space of the cell. Once the pipette is attached to the cell membrane, there are two methods of breaking the patch. The first is by applying more suction. The amount and duration of this suction depends on the type of cell and size of the pipette. The other method requires a large current pulse to be sent through the pipette. How much current is applied and the duration of the pulse also depend on the type of cell. This is referred to as the electrode . The pipette solution used usually approximates the high- potassium environment of the interior of the cell to minimize any changes this may cause. Generally speaking, there is a period at the beginning of a whole- cell recording, lasting approximately 1. In order: top- left, top- right, bottom- left, bottom- right. The name . After the whole- cell configuration is formed, the electrode is 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 convex membrane on the end of the electrode (like a ball open at the electrode tip), with the original outside of the membrane facing outward from the electrode. As the image at the right shows, this means that the fluid inside the pipette will be simulating the intracellular fluid, while a researcher is free to move the pipette and the bleb with its channels to another bath of solution. While multiple channels can exist in a bleb of membrane, single channel recordings are also possible in this conformation if the bleb of detached membrane is small and only contains one channel. The experimenter can perfuse the same patch with a variety of solutions in a relatively short amount of time, and if the channel is activated by a neurotransmitter or drug from the extracellular face, a dose- response curve can then be obtained. This ability to measure current through exactly the same piece of membrane in different solutions is the distinct advantage of the outside- out patch relative to the cell- attached method. On the other hand, it is more difficult to accomplish. The longer formation process involves more steps that could fail and results in a lower frequency of usable patches. Perforated patch. The main difference lies in the fact that when the experimenter forms the gigaohm seal, suction is not used 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 pores in the membrane, providing electrical access to the cell interior. 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, relative to whole- cell recordings, include the properties of the antibiotic pores, that allow equilibration only of small monovalent ions between the patch pipette and the cytosol, but not of larger molecules that cannot permeate through the pores. This property maintains endogenous levels of divalent ions such as Ca. AMP. Consequently, one can have recordings of the entire cell, as in whole- cell patch clamping, while retaining most intracellular signaling mechanisms, as in cell- attached recordings. As a result, there is reduced current rundown, and stable perforated patch recordings can last longer than one hour. This may decrease current resolution and increase recording noise. It can also take a significant amount of time for the antibiotic to perforate the membrane (about 1. B, and even longer for gramicidin and nystatin). 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. This technique was used as early as the year 1. Strickholm on the impedance of a muscle cell's surface. The closer the pipette gets to the membrane, the greater the resistance of the pipette tip becomes, but if too close a seal is formed, and it could become difficult to remove the pipette without damaging the cell. For the loose patch technique, the pipette does not get close enough to the membrane to form a gigaseal or a permanent connection, nor to pierce the cell membrane. This allows repeated measurements in a variety of locations on the same cell without destroying the integrity of the membrane. This flexibility has been especially useful to researchers for studying muscle cells as they contract under real physiological conditions, obtaining recordings quickly, and doing so without resorting to drastic measures to stop the muscle fibers from contracting.
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