Perforated Whole-Cell Patch-Clamp Recording
Perforated whole-cell patch-clamp is a variant of the patch-clamp technique used to measure the sum activity of ion channels in the plasma membrane of a single cell. Its defining feature is that electrical access to the cell is obtained through inclusion
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Introduction Conventional whole-cell patch-clamp electrophysiology involves sealing a glass micropipette (patch pipette) onto the surface of a single cell and subsequent rupture of the seal by sharp suction to obtain electrical access to the cell. The sum activity of all ion channels in the cell membrane can then be measured by voltage clamp. Using this technique, the experimenter has complete control over the cytosolic composition due to equilibration of the patch pipette solution with the cytosol. This allows Ca2+ to be buffered to a fixed value using EGTA or BAPTA and membrane impermeant drugs to be applied to the cytosol. However, conventional whole-cell patchclamp also results in washout of key endogenous cytosolic signalling molecules such as cAMP and Ca2+ resulting in the potential disruption of receptor-mediated signalling events. In 1988, Horn and Marty reported a new, less invasive method of obtaining electrical
Nikita Gamper (ed.), Ion Channels: Methods and Protocols, Methods in Molecular Biology, vol. 998, DOI 10.1007/978-1-62703-351-0_11, © Springer Science+Business Media, LLC 2013
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John E. Linley
access to a cell called the perforated-patch technique (1). In this technique, the sealed patch of membrane is perforated rather than ruptured by inclusion of a pore-forming antibiotic in the patch pipette. In this case, the equilibration between the pipette solution and cytosol is determined by the permeability properties of the antibiotic. Nystatin and amphotericin B are both commonly used antibiotics for perforated patch and have similar permeability properties, being permeable to small monovalent cations and anions such as Na+, K+, and Cl− and impermeable to large ions such as tetraethylammonium and cytosolic signalling molecules. The pore radius of nystatin is calculated to be ~0.4 nm (2), whereas amphotericin B forms a larger pore which is ~0.8 nm in radius (3), thereby excluding nonelectrolytes with a molecular weight >200. The cation selectivity sequence for both nystatin and amphotericin is Rb+ > K+ > Na+ > Li+, and is directly related to the size of the hydrated ions. Divalent cations such as Ca2+ and Mg2+ are impermeant. Chloride permeability of nystatin and amphotericin B is less than cation permeability (Panion/Pcation = 0.1) although Cl− still readily equilibrates in the perforated patch configuration. For electrophysiological recordings in which the endogenous intracellular Cl− concentration is desired to be maintained (for example when recording GABA responses in primary neurons), then the antibiotic gramicidin can be used. Gramicidin pores are permeable to small monovalent cations but impermeable to anions (4). However, gramicidin perforation is very slow compared to amphotericin B, typically taking ~30 min to stabilize, thereby impacting on the number of experiments which can be conducted in a day. Note that perforation is dependent on sterol composition of the lipid membrane (5). 1.1 Advantages of Perforated Patch
1. Minimal disruption of cytosolic components making it idea
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