Voltage Clamp Recordings from Xenopus Oocytes

Nathan Dascal1

1 Tel Aviv University, Ramat Aviv, Israel
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 6.12
DOI:  10.1002/0471142301.ns0612s10
Online Posting Date:  May, 2001
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Abstract

Xenopus oocytes serve as a standard heterologous expression system for the study of cloned ion channels. The large size of these cells allows for relatively easy expression and recording of activity of exogenous ion channels (together with neurotransmitter receptors and/or various regulatory proteins) using the whole-cell two-electrode voltage clamp (TEVC) technique, as well as standard single-channel patch clamp recordings. Although usually advantageous, the cell size also dictates certain limits on the accuracy of recordings and requires specific modifications of recording methods. However, combining the advantages of the system with available recording methods enables the use of Xenopus oocytes for sophisticated multidisciplinary studies of ion channels.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Two-Electrode Voltage Clamp (TEVC) Recording
  • Support Protocol: Fabrication of Agarose Cushion Microelectrodes (ACEs) and Agarose/KCl Bridges for TEVC
  • Basic Protocol 2: Patch Clamp Recordings from Oocytes
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Two-Electrode Voltage Clamp (TEVC) Recording

 Materials
  • 3 M KCl
  • Extracellular medium: ND96 (see recipe) and/or additional solutions (see recipes for high-Ba2+ solution and high-K+ solution)
  • TEVC apparatus (Axon Instruments, Dagan, NPI Electronic, Warner Instruments)
  • Two ACE microelectrodes (see Support Protocol)
  • Experimental chamber connected via two agarose/KCl bridges and Ag/AgCl wires to the electronic equipment as shown in Fig. 6.12.1 (also see Commentary and see Support Protocol)
  • Electrophysiological setup (see Strategic Planning and unit 6.1)
  • Oocytes injected with RNA (unit 4.3), placed in petri dishes with incubation solution
  • Pasteur pipet with an inner diameter slightly greater than that of the oocyte, thoroughly fire-polished to removed any sharp edges from the tip
  • Spiral shield made of silver or another metal with an inner diameter slightly greater than the electrode's

Support Protocol: Fabrication of Agarose Cushion Microelectrodes (ACEs) and Agarose/KCl Bridges for TEVC

 Materials
  • Agarose (molecular biology grade)
  • 3 M KCl and/or 3 M NaCl solutions (in H2O)
  • Standard glass tubes for preparation of microelectrodes (unit 6.3), soft or borosilicate glass, 1.6 to 2 mm o.d., 1.2 to 1.6 mm i.d.
  • Microelectrode puller (preferably vertical)
  • Microelectrode storage jar and a dry microelectrode storage stand (WPI).
  • 10-ml syringe with a 19- to 21-G needle
  • 10-ml syringe with a 17- to 19-G needle and a rubber or silicon tubing attached to the end of the needle, ~1 cm long, with i.d. slightly smaller than the o.d. of the glass tubes
  • Additional reagents and equipment for pulling microelectrodes (unit 6.3)

Basic Protocol 2: Patch Clamp Recordings from Oocytes

 Materials
  • Hypertonic solution 1 or 2 (see recipes)
  • Two 35-mm petri dishes filled with bath solution
  • Pasteur pipet with an inner diameter slightly greater than that of the oocyte, thoroughly fire-polished to remove any sharp edges from the tip
  • Dissecting binocular microscope with amplification up to 40×
  • Two pairs of fine (no. 55) forceps (it may be necessary to further sharpen the forceps on a fine sharpening stone under a dissecting microscope)
  • Experimental bath (constructed in-house; see Fig. 6.12.2) filled with bath solution
  • Standard patch clamp setup with an inverted microscope or (less appropriately) a dissecting binocular microscope with amplification of 50×
  • Additional reagents and equipment for patch-clamp recording (units 6.3-6.8)
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Figures

  •  FigureFigure 6.12.1 Principal scheme of a typical TEVC recording arrangement. Bold lines denote Ag/AgCl electrodes. The oocyte is placed in the middle of an experimental chamber filled with a recording solution (ND96). The level of solution is shown by dashed line. The experimental chamber is connected, via KCl/agarose bridges 1 and 2, to two compartments filled with 3 M KCl. The Ag/AgCl electrode of the left compartment is connected via bridge 1 to one of the inputs of the voltage-measuring amplifier built into the TEVC device. The second input of this amplifier (usually denoted as “voltage electrode” input on commercial TEVC devices) is connected via an Ag/AgCl electrode to the voltage recording glass microelectrode. The voltage measured by this device is fed into one of the inputs of the negative feedback amplifier, where it is compared with the Vcommand fed into the second input of this amplifier. Vcommand can be set manually by using a knob provided for this purpose on the front panel of the TEVC device, or input from an external source, typically driven by the computer. The output of the negative feedback amplifier (usually denoted as “current electrode” output on commercial TEVC devices) is connected to the current microelectrode via an Ag/AgCl electrode. The bath solution is also connected, via bridge 2, to the right 3 M KCl compartment, and from there, via an Ag/AgCl electrode, to the virtual ground circuit. The latter is supplied within a separate small box, or is built into the main body of the TEVC device. The outputs of the voltage-monitoring amplifier (measuring the membrane voltage, Vm) and of the virtual ground circuit (measuring the current flowing through the bath to the oocyte; the membrane current, Im) are connected to a computer and (optionally) additional equipment such as VCR, oscilloscope, or chart recorder. The connections to the computer and additional equipment are not shown here. Commercially available TEVC devices always have special outputs for monitoring Vm and Im that are used to make these connections.
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  •  FigureFigure 6.12.2 The recommended oocyte perfusion experimental chamber. (A) general view; (B) view from above; (C) front view on a longitudinal section made along the median line of the oocyte compartment. Dimensions are given in mm. The chamber is made of Perspex. The oocyte compartment (a) is 2 mm deep, 2 mm wide, and ~12 mm long and has a cavity in the middle of the compartment's floor, ~0.2 mm deep and 0.3 to 0.4 mm in diameter. The diameters of all tubes drilled within the body of the chamber (c, f, g) is 1.2 or 1.3 mm. The KCl compartments (d, e) are 7 mm in diameter and 5 mm deep. These and other dimensions are indicated in the drawing. See Background Information for further detailed discussion.
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  •  FigureFigure 6.12.3 A simplified electrical scheme of a TEVC setup. The resistances of voltage and current electrodes (Re1 and Re2) and KCl/agarose bridges (Rbridge1 and Rbridge2) are chosen under the assumption that the electrodes and the bridges have been made according to recommendations in the text. The resistance and capacitance of the resting oocyte membrane (Rm and Cm, respectively) and of the cytosol (Rcyt) are typical for a fully grown defolliculated oocyte of a 1 to 1.2 mm size.
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Literature Cited

 Literature Cited
    Attali, B., Latter, H., Rachamim, N., and Garty, H. 1995. A corticosteroid-induced gene expressing an “IsK-like” K+ channel activity in Xenopus oocytes. Proc. Natl. Acad. Sci. U.S.A. 92:6092-6096.
    Barnard, E.A., Miledi, R., and Sumikawa, K. 1982. Translation of exogenous messenger RNA coding for nicotinic acetylcholine receptors produces functional receptors in Xenopus oocytes. Proc. R. Soc. Lond. B. Biol. Sci. 215:241-246.
    Bezanilla, F., Perozo, E., Papazian, D.M., and Stefani, E. 1991. Molecular basis of gating charge immobilization in Shaker potassium channels. Science 254:679-683.
    Boton, R., Dascal, N., Gillo, B., and Lass, Y. 1989. Two calcium-activated chloride conductances in Xenopus laevis oocytes permeabilized with the ionophore A23187. J. Physiol. (Lond.) 408:511-534.
    Conti, F. and Stuhmer, W. 1989. Quantal charge redistributions accompanying the structural transitions of sodium channels. Eur. Biophys. J. 17:53-59.
    Costa, A.C., Patrick, J.W., and Dani, J.A. 1994. Improved technique for studying ion channels expressed in Xenopus oocytes, including fast superfusion. Biophys. J. 67:395-401.
    Dascal, N. 1987. The use of Xenopus oocytes for the study of ion channels. CRC Crit. Rev. Biochem. 22:317-387.
    Dascal, N. and Lotan, I. 1991. Activation of protein kinase C alters voltage dependence of a Na+ channel. Neuron 6:165-175.
    Dascal, N. and Lotan, I. 1992. Expression of exogenous ion channels and neurotransmitter receptors in RNA-injected Xenopus oocytes. In Protocols in Molecular Neurobiology, Chapter 13. pp. 205-225. Humana Press, Totowa, N.J.
    Dascal, N., Lim, N.F., Schreibmayer, W., Wang, W., Davidson, N., and Lester, H.A. 1993. Expression of an atrial G-protein-activated potassium channel in Xenopus oocytes. Proc. Natl. Acad. Sci. U.S.A. 90:6596-6600.
    Dascal, N., Lotan, I., Karni, E., and Gigi, A. 1992. Calcium channel currents in Xenopus oocytes injected with rat skeletal muscle RNA. J. Physiol. (Lond.) 450:469-490.
    Finkel, A.S. and Cage, P.W. 1985. Conventional voltage clamping with two intracellular microelectrodes. In Voltage and Patch Clamping with Microelectrodes.( T. Smith, Jr., et al., eds.) Williams & Wilkins, Baltimore.
    Hamill, O.P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F.J. 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391:85-100.
    Hamill, O.P. and McBride, D.W. Jr. 1996. The pharmacology of mechanogated membrane ion channels. Pharmacol Rev. 48:231-252.
    Hartzell, H.C. 1996. Activation of different Cl currents in Xenopus oocytes by Ca liberated from stores and by capacitative Ca influx. J. Gen. Physiol. 108:157-175.
    Hedin, K.E., Lim, N.F., and Clapham, D.E. 1996. Cloning of a Xenopus laevis inwardly rectifying K+ channel subunit that permits GIRK1 expression of IKACh currents in oocytes. Neuron 16:423-429.
    Hilgemann, D.W. and Lu, C.C. 1998. Giant membrane patches: Improvements and applications. Methods Enzymol. 293:267-280.
    Liman, E.R., Tytgat, J. and Hess, P. 1992. Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs. Neuron 9:861-871.
    Madeja, M., Musshoff, U., and Speckmann, E.J. 1991. A concentration-clamp system allowing two-electrode voltage-clamp investigations in oocytes of Xenopus laevis. J. Neurosci. Methods. 38:267-269.
    Methfessel, C., Witzemann, V., Takahashi, T., Mishina, M., Numa, S., and Sakmann, B. 1986. Patch clamp measurements on Xenopus laevis oocytes: Currents through endogenous channels and implanted acetylcholine receptor and sodium channels. Pflugers Arch. 407:577-588.
    Miledi, R. 1982. A calcium-dependent transient outward current in Xenopus laevisoocytes. Proc. R. Soc. Lond. B. 215:491-497.
    Schreibmayer, W., Lester, H.A., and Dascal, N. 1994. Voltage clamp of Xenopus laevis oocytes utilizing agarose cushion electrodes. Pflugers Arch. 426:453-458.
    Taglialatela, M., Toro, L., and Stefani, E. 1992. Novel voltage clamp to record small, fast currents from ion channels expressed in Xenopus oocytes. Biophys. J. 61:78-82.
    Takahashi, T., Neher, E., and Sakmann, B. 1987. Rat brain serotonin receptors in Xenopus oocytes are coupled by intracellular calcium to endogenous channels. Proc. Natl. Acad. Sci. U.S.A. 84:5063-5067.
    Vasilets, L.A., Schmalzing, G., Madefessel, K., Haase, W., and Schwarz, W. 1990. Activation of protein kinase C by phorbol ester induces downregulation of the Na+/K+-ATPase in oocytes of Xenopus laevis. J. Membr. Biol. 118:131-142.
    White, M.M. and Aylwin, M. 1990. Niflumic and flufenamic acids are potent reversible blockers of Ca2+-activated Cl– channels in Xenopus oocytes. Mol. Pharmacol. 37:720-724.
    Zhang, Y., McBride, D.W., Jr., and Hamill, O.P. 1998. The ion selectivity of a membrane conductance inactivated by extracellular calcium in Xenopus oocytes. J. Physiol. (Lond.) 508:763-776.
 Key References
    Schreibmayer et al., 1994. See above.

Original paper describing the fabrication of ACEs and series resistance compensation in a circuit without virtual ground.

    Stuhmer, W. 1998. Electrophysiologic recordings from Xenopus oocytes. Methods Enzymol. 293:280-300.

A valuable methodological manual of oocyte electrophysiology.

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