Hamill81

Hamill81 - Pflfigers Arch (1981)391:85-100 Pfltigers Archiv...

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Pflfigers Arch (1981)391:85-100 Pfltigers Archiv European Journal of Physiology Springer-Verlag 1981 Improved Patch-Clamp Techniques for High-Resolution Current Recording from Cells and Cell-Free Membrane Patches O. P. Hamill, A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth Max-Planck-Institut ffir biophysikalische Chemic, Postfach 968, Am Fassberg, D-3400 G6ttingen, Federal Republic of Germany Abstract. 1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further:~efined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabri- cation of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette- membrane seals with resistances of 10 9- 1011 ~. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time re- solution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (< 20 gin). 7. The wide range of cell types amenable to giga-seal formation is discussed. Key words: Voltage-clamp - Membrane currents - Single channel recording - Ionic channels Introduction The extracellular patch clamp technique has allowed, for the first time, the currents in single ionic channels to be observed (Neher and Sakmann 1976). In this technique a small heat- polished glass pipette is pressed against the cell membrane, forming an electrical seal with a resistance of the order of 50 Mr2 (Neher et al. 1978). The high resistance of this seal ensures that most of the currents originating in a small patch Send offprint requests to B. Sakmann at the above address of membrane flow into the pipette, and from there into current-measurement circuitry. The resistance of the seal is important also because it determines the level of background noise in the recordings. Recently it was observed that tight pipette-membrane seals, with resistances of 10-100 Gf2, can be obtained when
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Hamill81 - Pflfigers Arch (1981)391:85-100 Pfltigers Archiv...

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