Cut-open oocyte voltage-clamp technique.
In this article, we described the use and limitations of the COVG technique. The advantages of the present method as follows: 1. High-frequency response and low noise recording (24-microseconds time constant and 1.2-nA rms at 5 kHz). This allows the accurate description of small gating currents and fast activation or deactivation of ionic currents to be adequately resolved. Currents up to 20-30 microA can be adequately clamped. 2. Stable recording conditions lasting for several hours. Even though this technique has internal access, rundown is minimized compared to excised patches. 3. Access to the cell interior. The intracellular medium can be exchanged with various solutions. This was of invaluable help in recording K+ gating currents, an experimental condition that required the complete blockade of all ionic currents. This advantage will make the present method suitable for the study of channel modulation by second messengers and drugs. In addition, channel selectivity properties can be defined by ion substitution experiments. 4. Channel localization. A study of the membrane compartmentalization of channels can be easily obtained with this method, since it allows the voltage clamping of different regions of the oocyte surface.
Department of Anesthesiology, University of California, Los Angeles 90095, USA.
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Stefani
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Bezanilla
D000818:Animals; D015220:Calcium Channels / Q000096:biosynthesis / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology; D002463:Cell Membrane Permeability; D003001:Cloning, Molecular / Q000379:methods; D004594:Electrophysiology / Q000379:methods*; D004867:Equipment Design; D005260:Female; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D008564:Membrane Potentials / Q000502:physiology; D016296:Mutagenesis; D009865:Oocytes / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000295:instrumentation*; D015221:Potassium Channels / Q000096:biosynthesis / Q000502:physiology*; D018075:RNA, Complementary / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D012503:Saponins; D017384:Sequence Deletion; D051637:Shaker Superfamily of Potassium Channels; D013997:Time Factors; D014982:Xenopus laevis
D015220:Calcium Channels; D015221:Potassium Channels; D018075:RNA, Complementary; D011994:Recombinant Proteins; D012503:Saponins; D051637:Shaker Superfamily of Potassium Channels