Patch clamp

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Patch clamp of a nerve cell. The pipette in the photograph has been marked with a slight blue color.
A bacterial spheroplast patched with a glass pipette

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]

Basic technique[edit]

Set-up[edit]

Patch clamp recording uses, as a recording electrode, a glass micropipette often called a patch pipette, and another electrode in the bath around the cell. 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.[2] This small size is used to enclose a membrane surface area or "patch" that often contains just one or a few ion channel molecules.[3] This type of electrode is distinct from the "sharp microelectrode" used to puncture cells in traditional intracellular recordings, in that it is sealed onto the surface of the cell membrane, rather than inserted through it.

In some experiments, the micropipette tip is heated in a microforge to produce a smooth surface that assists in forming a high resistance seal with the cell membrane. 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 semivesicle which, if formed properly, creates a resistance in the 10-100 gigaohms.[3] The seal created is called a "gigaohm seal" or "gigaseal," since the electrical resistance of that seal is in excess of a gigaohm. The high resistance of this seal makes it possible to isolate electronically the currents measured across the membrane patch with little competing noise, as well as providing some mechanical stability to the recording.[4]

Depending on the experiment, the interior of the pipette can be filled with a solution matching the ionic composition of the bath solution, as in the case of cell-attached recording, or the cytoplasm for whole-cell recording. The researcher can also change the content or concentration of this solution by adding ions or drugs to study the ion channels under different conditions.

A patch clamp recording of current reveals transitions between two conductance states of a single ion channel: closed (at top) and open (at bottom), as well as a several "intermediate steps".

Recording[edit]

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. To make these recordings, the patch pipette is compared to a bath electrode. Current is then injected into the system to maintain a constant voltage. However much current is needed to clamp the voltage is opposite of the current through the membrane. [3]

Alternatively, the cell can be current clamped in whole-cell mode, keeping current constant while observing changes in membrane voltage.

Variations[edit]

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 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. 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[edit]

Cell-attached Patch Clamp

For this method the pipette is sealed onto the cell membrane to obtain a gigaseal, while ensuring that the cell membrane remains intact. By only attaching to the exterior of the cell membrane, there is very little disturbance to the structure and environment of the cell.[3] Also by not disrupting the interior of the cell, any intracellular activity normally influenced by the channel will still be able to function as it would physiologically.[5] This allows the recording of currents through single, or sometimes multiple, ion channels contained in the patch of membrane captured by the pipette. Using this method it is also relatively easy to obtain the right configuration and once obtained it is fairly stable.[6]

For ligand-gated ion channels or channels that are modulated by metabotropic receptors, the neurotransmitter or drug being studied is usually included in the pipette solution, where it can interact with what used to be the external surface of the membrane. The resulting channel activity can be attributed to the drug being used. Although it is usually 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 using several cells and patches. However, voltage-gated ion channels can be clamped at different membrane potentials using the same patch. This results in graded channel activation, and a complete I-V (current-voltage) curve can be established with only one patch. Another potential drawback of this technique is the limited access to the inside of the cell. It can only be indirectly altered and its exact composition is not known.[6]

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.[7] 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]

Excised Inside-Out Patch Clamp

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.[8] 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.[9]

Whole-cell recording or whole-cell patch[edit]

Whole-cell patch clamp

Whole-cell recordings involve recording currents through multiple channels simultaneously, over the membrane of the entire cell. The electrode is left in place on the cell, but more suction is applied to rupture the membrane patch, thus providing access 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 applied and the duration of the pulse depend on the type of cell.[6]

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. There is also a large input resistance which allows for clearer measurements.[10] A disadvantage of this technique is that because the volume of the electrode is larger than the volume of the cell, 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.[6] After a while, 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 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 10 minutes, when one can take measurements before the cell has been dialyzed.

Outside-out patch[edit]

Outside-out patch clamp formation technique. In order: top-left, top-right, bottom-left, bottom-right

The name "outside-out" seems to have been coined in a 1981 paper by Hamill, et al on Improved Patch-Clamp Techniques. It emphasizes both this technique's similarity to the inside-out technique and the fact that it places the external surface of the cell membrane and its channels on the outside of the vessel formed by the glass electrode (labeled "pipette" in the image at right) and the patch of membrane.[5]

The formation of an outside-out patch begins with a whole-cell patch. After the seal 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 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.[5] As the graphic at the right shows, this means that the fluid inside the pipette will be simulating 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.[11] The picture at right illustrates the second of these cases.

Outside-out patching gives the experimenter the opportunity to examine the properties of an ion channel when it is isolated from the cell and exposed to different solutions on the extracellular surface of the membrane. 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 from the extracellular face, a dose-response curve can then be obtained.[12] This ability to measure current through the exact 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[edit]

Perforated Patch Clamp

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.[13] 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.[13]

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.[13]

Loose patch[edit]

Loose patch clamp technique

Loose patch clamp is different from the other techniques discussed here in that it employs a loose seal (low electrical resistance) rather than the tight gigaseal used in the conventional technique. This kind of technique was used as early as the year 1961, as described in a paper by Strickholm on the impedance of a muscle cell's surface,[14] but received little attention until being brought up again and given a name by Almers, Stanfield, and Stühmer in 1982[15]—after patch clamps had been identified as a major tool of electrophysiology.

To achieve a loose patch clamp on a cell membrane, the pipette is pushed toward the cell slowly, until the electrical resistance of the seal between the muscle cell and the pipette increases to a few times greater resistance than that of the electrode. The closer the pipette gets to the membrane, the greater the resistance of the seal becomes, but if too strong a seal is formed, 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 push hard against it and form a gigaseal or a permanent connection, nor to pierce the cell membrane.[16] Notice in the image at right that the membrane is intact, and that the lack of a tight seal creates a gap through which ions can pass, though not as easily as moving down the pipette.

A significant advantage of the loose seal is that the pipette that is used can be repeatedly removed from the membrane after recording, and the membrane will remain intact. 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 under real physiological conditions, obtaining recordings quickly, and doing so without resorting to drastic measures to stop the muscle fibers from contracting[15] A major disadvantage is that the resistance between the pipette and the membrane is greatly reduced, allowing current to leak through the seal. This leakage can be corrected for, however, which offers the opportunity to compare and contrast recordings made from different areas on the cell of interest. Given this, it has been estimated that the loose patch technique can resolve currents smaller than 1 mA/cm2.[16]

Automatic patch clamping[edit]

Automated patch clamp systems have recently been developed, in order to collect large amounts of data inexpensively in a shorter period of time. Such systems typically include a single-use microfluidic device, either an injection molded or a PDMS cast chip, to capture a cell or cells, and an integrated electrode.

See also[edit]

References[edit]

  1. ^ "The Nobel Prize in Physiology or Medicine 1991". nobelprize.org. Nobel Media AB. Retrieved November 8, 2014. 
  2. ^ Bannister, Niel (November 1, 2012). Langton, Phil, ed. Essential Guide to Reading Biomedical Papers: Recognizing and Interpreting Best Practice. Wiley-Blackwell. ISBN 9781118402184. 
  3. ^ a b c d Sakmann, B.; Neher, E. (1984). "Patch clamp techniques for studying ionic channels in excitable membranes". Annual Reviews Physiology 46: 455–472. Retrieved November 10, 2014. 
  4. ^ Sigworth, Fredrick J.; Neher, E. (October 2, 1980). "Single Na+ channel currents observed in cultured rat muscle cells". Nature 287: 447–449. 
  5. ^ a b c Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. (1981). "Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches". Pflügers Archiv European Journal of Physiology 391 (2): 85–100. doi:10.1007/BF00656997. PMID 6270629. 
  6. ^ a b c d Molleman, Areles (March 6, 2003). Patch Clamping: An Introductory Guide To Patch Clamp Electrophysiology. Wiley. ISBN 9780470856529. Retrieved November 2, 2014. 
  7. ^ Veitinger, Sophie. "The Patch-Clamp Technique". Science Lab. Leica Microsystems. Retrieved November 10, 2014. 
  8. ^ Ogden, David; Stanfield, Peter. "Patch Clamp Techniques" (pdf). utdallas.edu. pp. 53–78. Retrieved November 11, 2014. 
  9. ^ 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. 
  10. ^ Staley, K.J. (May 1, 1992). "Membrane properties of dentate gyrus granule cells: comparison of sharp microelectrode and whole-cell recordings". Journal of Neurophysiology 67 (5): 1346–1358. 
  11. ^ Howe, JR; Cull-Candy, SG; Colquhoun, D (Jan 1991). "Currents through single glutamate receptor channels in outside-out patches from rat cerebellar granule cells". Journal of Physiology 432 (1): 143–202. PMID 1181322. Retrieved 11 November 2014. 
  12. ^ von Beckerath, N; Adelsberger, H; Parzefall, F; Franke, C; Dudel, J (Apr 1995). "GABAergic inhibition of crayfish deep extensor abdominal muscle exhibits a steep dose-response relationship and a high degree of cooperativity". European Journal of Physiology 429 (6): 781–788. PMID 7541524. Retrieved 11 November 2014. 
  13. ^ a b c Linley, John (2013). "11". In Gamper, Nikita. Perforated Whole-Cell Patch-Clamp Recording (Second ed.). Humana Press. pp. 149–157. ISBN 978-1-62703-351-0. Retrieved November 10, 2014. 
  14. ^ Strickholm, A (1 Jul 1961). "Impedance of a Small Electrically Isolated Area of the Muscle Cell Surface". Journal of General Physiology 44 (6): 1073. PMID 19873540. Retrieved 13 November 2014. 
  15. ^ a b Almers W, Stanfield PR, Stühmer W. (1983). "Lateral distribution of sodium and potassium channels in frog skeletal muscle: measurements with a patch clamp method". Journal of Physiology 336: 261–284. PMID 1198969. 
  16. ^ a b Lupa, MT; Caldwell, JH (Nov 1991). "Effect of Agrin on the Distribution of Acetylcholine Receptors and Sodium Channels on Adult Skeletal Muscle Fibers in Culture". Journal of Cell Biology 115 (3): 765–778. PMID 1655812. Retrieved 10 November 2014. 

External links[edit]

Category:Neurophysiology Category:Physiology Category:Electrophysiology Category:Laboratory techniques