Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches
- 9k Downloads
The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches.
A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipettemembrane seals with resistances of 109–1011Ω.
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.
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 resolution achieved with giga-seals.
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.
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 (<20μm).
The wide range of cell types amenable to giga-seal formation is discussed.
Key wordsVoltage-clamp Membrane currents Single channel recording Ionic channels
Unable to display preview. Download preview PDF.
- Brandt BL, Hagiwara S, Kidokoro Y, Miyazaki S (1976) Action potentials in the rat chromaffin cell and effects of Acetylcholine. J Physiol (Lond) 263:417–439Google Scholar
- Cass A, Dalmark M (1973) Equilibrium dialysis of ions in nystatintreated red cells. Nature (New Biol) 244:47–49Google Scholar
- Fenwick EM, Fajdiga PB, Howe NBS, Livett BG (1978) Functional and morphological characterization of isolated bovine adrenal medullary cells. J Cell Biol 76:12–30Google Scholar
- Gage PW, Van Helden D (1979) Effects of permeant monovalent cations on end-plate channels. J Physiol (Lond) 288:509–528Google Scholar
- Hamill OP, Sakmann B (1981) A cell-free method for recording single channel currents from biological membranes. J Physiol (Lond) 312:41–42PGoogle Scholar
- Horn R, Brodwick MS (1980) Acetylcholine-induced current in perfused rat myoballs. J Gen Physiol 75:297–321Google Scholar
- Horn R, Patlak JB (1980) Single channel currents from excised patches of muscle membrane. Proc Natl Acad Sci USA 77:6930–6934Google Scholar
- Kostyuk PG, Krishtal OA (1977) Separation of sodium and calcium currents in the somatic membrane of mollusc neurones. J Physiol (Lond) 270:545–568Google Scholar
- Kostyuk PG, Krishtal OA, Pidoplichko VI (1976) Effect of internal fluoride and phosphate on membrane currents during intracellular dialysis of nerve cells. Nature 257:691–693Google Scholar
- Krishtal OA, Pidoplichko VI (1975) Intracellular perfusion of Helix neurons. Neurophysiol (Kiev) 7:258–259Google Scholar
- Krishtal OA, Pidoplichko VI (1980) A receptor for protons in the nerve cell membrane. Neuroscience 5:2325–2327Google Scholar
- Läuger P (1975) Shot noise in ion channels. Biochim Biophys Acta 413:1–10Google Scholar
- Langmuir I (1938) Overturning and anchoring of monolayers. Science 87:493–500Google Scholar
- Lee KS, Akaike N, Brown AM (1978) Properties of internally perfused, voltage clamped, isolated nerve cell bodies. J Gen Physiol 71:489–508Google Scholar
- Neher E (1981) Unit conductance studies in biological membranes. In: Baker PF (ed), Techniques in cellular physiology, Elsevier/North-Holland, AmsterdamGoogle Scholar
- Neher E, Sakmann B (1976) Single channel currents recorded from membrane of denervated frog muscle fibres. Nature 260:799–802Google Scholar
- Neher E, Sakmann B, Steinbach JH (1978) The extracellular patch clamp: A method for resolving currents through individual open channels in biological membranes. Pflügers Arch 375:219–228Google Scholar
- Nir S, Bentz J (1978) On the forces between phospholipid bilayers. J Colloid Interface Sci 65:399–412Google Scholar
- Parsegian VA, Fuller N, Rand RP (1979) Measured work of deformation and repulsion of lecithin bilayers. Proc Natl Acad Sci USA 76: 2750–2754Google Scholar
- Petrov JG, Kuhn H, Möbius D (1980) Three-Phase Contact Line Motion in the deposition of spread monolayers. J Colloid Interface Sci 73:66–75Google Scholar
- Sakmann B, Patlak J, Neher E (1980) Single acetylcholine-activated channels show burst-kinetics in presence of desensitizing concentrations of agonist. Nature 286:71–73Google Scholar
- Sigworth FJ, Neher E (1980) Single Na+ channel currents observed in cultured rat muscle cells. Nature 287:447–449Google Scholar
- Stevens CF (1972) Inferences about membrane properties from electrical noise measurements. Biophys J 12:1028–1047Google Scholar