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[1]. This small size is used to enclose a membrane surface area or "patch" that often contains just one or a few ion channel molecules.[2] 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.[2]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.[3]
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.
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. [2]
Alternatively, the cell can be current clamped in whole-cell mode, keeping current constant while observing changes in membrane voltage.
Cell-attached Patch edit
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.[2] 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.[4]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.[5]
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.[5]
Whole-cell recording or whole-cell patch edit
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.[5]
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. [6] 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.[5] 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.
See also edit
References edit
- ^ Bannister, Niel (November 1, 2012). Langton, Phil (ed.). Essential Guide to Reading Biomedical Papers: Recognizing and Interpreting Best Practice. Wiley-Blackwell. ISBN 9781118402184.
- ^ 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.
- ^ Sigworth, Fredrick J.; Neher, E. (October 2, 1980). "Single Na+ channel currents observed in cultured rat muscle cells". Nature. 287: 447–449.
- ^ 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.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - ^ 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.
- ^ 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.
- Kandel E.R., Schwartz, J.H., Jessell, T.M. (2000). Principles of Neural Science, 4th ed. McGraw-Hill, New York. ISBN 978-0-8385-7701-1.
{{cite book}}: CS1 maint: multiple names: authors list (link) pp. 152–153. - Tanzi S, Østergaard PF, Matteucci M, Christiansen TL, Cech J, Marie R, Taboryski RJ (2012). "Fabrication of combined-scale nano- and microfluidic polymer systems using a multilevel dry etching, electroplating and molding process". Journal of Micromechanics and Microengineering. 22 (11): 115008. Bibcode:2012JMiMi..22k5008T. doi:10.1088/0960-1317/22/11/115008.
{{cite journal}}: CS1 maint: multiple names: authors list (link)
External links edit
Category:Neurophysiology Category:Physiology Category:Electrophysiology Category:Laboratory techniques