Skip to main content
SpringerLink
Log in

The Patch-Clamp Technique for Measurement of K+Channels in Xenopus Oocytes and Mammalian Expression Systems

  • Chapter
Potassium Channels in Cardiovascular Biology

  • 227 Accesses

Abstract

Potassium channels are ubiquitous membrane proteins. They are expressed in most cells, both excitable and nonexcitable. In excitable cells, K+ channels preside over a wide range of cell functions such as excitability, propagation of excitation, and neurotransmitter release (Hille, 1992; Rudy, 1988). In nonexcitable cells, they are responsible for regulating cell volume and cell proliferation (Sobko et al., 1998; Hille, 1992). Therefore, most cells express different voltage-dependent K+ (Kv) channels to guarantee certain functions and to optimize the cellular response to different stimuli (Sobko et al., 1998; Conforti and Millhorn, 1997). The heteromultimeric nature of native Kv channels and the possibility that several related types of Kv channels of different compositions are expressed in the same cell complicate the study of an isolated channel in mammalian cells (Shamotienko et al., 1997; Sheng et al., 1993; Wang et al., 1993). Study of the behavior of a particular type of K+ channel in the native cell is further complicated by the limited selectivity of available K+ channel blockers and toxins (Hopkins, 1998; Grissmer et al., 1994). Kv channels of known composition can be expressed in heterologous expression systems, such as the Xenopus oocytes and mammalian cell lines. Structural and functional studies of Kv channels in expression systems provide important information for understanding, by extrapolation, the function of similar channels in native cells.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Similar content being viewed by others

References

  • Ahring, P. K., Strobaek, D., Christophersen, P., Olesen, S. P., and Johansen, T. E., 1997, Stable expression of the human large-conductance Ca2+-activated K+ channel alpha- and beta-subunits in HEK293 cells, FEBS Lett. 415:67–70.

    Article  PubMed  CAS  Google Scholar 

  • Ammala, C., Moorhouse, A., and Ashcroft, F. M., 1996, The sulphonylurea receptor confers diazoxide sensitivity on the inwardly rectifying K+ channel Kir6.1 expressed in human embryonic kidney cells, J. Physiol. (London) 494:709–714.

    CAS  Google Scholar 

  • Berjukow, S., Doring, F., Froschmayr, M., Grabner, M., Glossman, H., and Hering, S., 1996, Endogenous calcium channels in human embryonic kidney (HEK293) cells, Br. J. Pharmacol 118:748–754.

    Article  PubMed  CAS  Google Scholar 

  • Chabala, L. D., Bakry, N., and Covarrubias, M., 1993, Low molecular weight poly(A)+ mRNA species encode factors that modulate gating of a non-Shaker A-type K+ channel, J. Gen. Physiol. 102:713–728.

    Article  PubMed  CAS  Google Scholar 

  • Chalfie, M., Euskirchen, G., Ward, W. W., and Prasher, D. C., 1994, Green fluorescent protein as a marker for gene expression, Science 263:802–805.

    Article  PubMed  CAS  Google Scholar 

  • Choe, H., and Sackin, H., 1997, Improved preparation of Xenopus oocytes for patch-clamp recording, Pflügers Arch. 433:648–652.

    Article  PubMed  CAS  Google Scholar 

  • Chomczynski, P., and Sacchi, N., 1987, Single step method for RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem. 162:156–159.

    Article  PubMed  CAS  Google Scholar 

  • Claudio, T, 1992, Stable expression of heterologous multisubunit protein complexes established by calcium phosphate- or lipid-mediated co-transfection, Methods Enzymol. 207:391–408.

    Article  PubMed  CAS  Google Scholar 

  • Conforti, L., and Millhorn, D. E., 1997, Selective inhibition of a slow-inactivating voltage-dependent K+ channel in rat PC12 cells by hypoxia, J. Physiol. (London) 502:293–305.

    Article  CAS  Google Scholar 

  • Covarrubias, M., Wei, A., and Salkoff, L., 1991, Shaker, Shal, Shab and Shaw express independent K+ current systems, Neuron 5:847–856.

    Google Scholar 

  • Dumont, J. N., 1972, Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals, J. Morphol. 136:153–179.

    Article  PubMed  CAS  Google Scholar 

  • Frech, G. C., VanDongen, A. M. J., Schuster, G., Brown, A. M., and Joho, R. H., 1989, A novel potassium channel with delayed rectifier properties isolated from rat brain by expression cloning, Nature 340:642–645.

    Article  PubMed  CAS  Google Scholar 

  • Goldin, A. L., 1992, Maintenance of Xenopus laevis and oocyte injection, Methods Enzymol. 207:266–297’.

    Article  PubMed  CAS  Google Scholar 

  • Grissmer, S., Nguyen, A. N., Aiyar J., Hanson, D. C., Mather, R. J., Gutman, G. A., Karmilowicz, M. J., Auperin, D. A., and Chandy, K. G., 1994, Pharmacological characterization of five cloned voltage-gated K+ channels, type Kv1.1, 1.2,1.2,1.5 and 3.1, stably expressed in mammalian cell lines, Mol. Pharmacol. 45:1227–1234.

    PubMed  CAS  Google Scholar 

  • 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, Pflügers Arch. 391:85–100.

    Article  PubMed  CAS  Google Scholar 

  • Hart, P. J., Overturf, K. E., Russel, S. N., Carl, A., Hume, J. R., Sanders, K. M., and Horowitz, B., 1993, Cloning and expression of a Kv1.2 class delayed rectifier K+ channel from canine smooth muscle, Proc. Natl. Acad. Sct. U.S.A. 90:9659–9663.

    Article  CAS  Google Scholar 

  • Heim, R., and Tsien, R. Y., 1996, Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer, Curr. Biol. 6(2):178–182.

    Article  PubMed  CAS  Google Scholar 

  • Heim, R., Prasher, D. C., and Tsien, R. Y., 1994, Wavelength mutations and posttranslational autoxidation of green fluorescent protein, Proc. Natl. Acad. Sci. U.S.A. 91:12501–12504.

    Article  PubMed  CAS  Google Scholar 

  • Heinemann, S. H., Rettig, J., Graack, H.-R., and Pongs, O., 1996, Functional characterization of Kv channel β-subunits from rat brain, J. Physiol. (London) 493:625–633.

    CAS  Google Scholar 

  • Hilgemann, D. W., 1995, The giant membrane patch, in: Single-Channel Recording, 2nd Ed. (B. Sakmann E. Neher eds.), Plenum Press, New York, pp. 307–327.

    Google Scholar 

  • Hille, B., 1992, Potassium channels and chloride channels, in: Ion Channels of Excitable Membranes, 2nd ed. Sinauer Associates, Sunderland, Massachusetts, pp. 115–139.

    Google Scholar 

  • Honore, E., Attali, B., Romey, G., Lesage, F., Barhanin, J., and Lazdunski, M., 1992, Different types of K+ channel current are generated by different levels of a single mRNA, EMBO J. 11:2465–2471.

    PubMed  CAS  Google Scholar 

  • Hopkins, W. F., 1998, Toxin and subunit specificity of blocking affinity of three peptide toxins for heteromultimeric, voltage-gated potassium channels expressed in Xenopus oocytes, J. Pharmacol. Exp. Ther. 285:1051–1060.

    PubMed  CAS  Google Scholar 

  • Ikeda, S. R., Soler, F., Zuhlke, R. D., Joho, R. H., and Lewis, D. L., 1992, Heterologous expression of the human potassium channel Kv2.1 in clonal mammalian cells by direct cytoplasmic microinjection of cRNA, Nature 422:201–203.

    CAS  Google Scholar 

  • Jan, L. Y., and Jan, Y. N., 1992, Structural elements involved in specific K+ channel functions, Annu. Rev. Physiol. 54:537–555.

    Article  PubMed  CAS  Google Scholar 

  • John, S. A., Monck, J. R., Weiss, J. N., and Ribalet, B., 1998, The sulphonylurea receptor SUR1 regulates ATP-sensitive mouse Kir6.2 K+ channels linked to the green fluorescent protein in human embryonic kidney cells (HEK 293), J. Physiol. (London) 510:333–345.

    Article  CAS  Google Scholar 

  • Karschin, A., Thorne, B. A., Thomas, G., and Lester, H. A., 1992, Vaccina virus vector to express ion channel genes, Methods Enzymol. 207:408–423.

    Article  PubMed  CAS  Google Scholar 

  • Kramer, R. H., 1990, Patch cramming: Monitoring intracellular messengers in intact cells with membrane patches containing detector ion channels, Neuron 2:335–341.

    Article  Google Scholar 

  • Lalik, P. H., Krafte, D. S., Volberg, W. A., and Ciccarelli, R. B., 1993, Characterization of endogenous sodium channels gene expressed in Chinese hamster ovary cells, Am. J. Physiol. 264:C803–C809.

    PubMed  CAS  Google Scholar 

  • Levitan, B., 1999, Tagging potassium channels with fluorescent protein to study mobility and interactions with other proteins, Methods Enzymol. 294:47–59.

    Article  PubMed  CAS  Google Scholar 

  • Li, M., Jan Y. N., and Jan, L. Y., 1992, Specification of subunit assembly by the hydrophilic amino-terminal domain of the Shaker potassium channel, Science 257:1225–1230.

    Article  PubMed  CAS  Google Scholar 

  • Lindau, M., and Fernandez, J. M., 1986, Patch-clamp study of histamine secreting cells, J. Gen. Physiol. 88:349–368.

    Article  PubMed  CAS  Google Scholar 

  • Lory, P., Rassendren, F. A., Richard, S., Tiaho, F., and Nargeot, J., 1990, Characterization of voltage-dependent calcium channels expressed in Xenopus oocytes injected with mRNA from rat heart, J Physiol. (London) 429:95–112.

    CAS  Google Scholar 

  • Lu, L., Montrose-Rafizadeh, C., Hwang, T. C., and Guggino, W. B., 1990, A delayed rectifier potassium current in Xenopus oocytes, Biophys. J. 57:1117–1123.

    Article  PubMed  CAS  Google Scholar 

  • Lupu-Meiri, M., Shapira, H., and Oron, Y., 1988, Hemispheric asymmetry of rapid chloride responses to inositol triphosphate and calcium in Xenopus oocytes, FEBS Lett. 240:8387.

    Article  Google Scholar 

  • Marshall, J., Molloy, R., Moss, G. W. J., Howe, J. R. and Hughes, T. E., 1995, The jellyfish green fluorescent protein: A new tool for studying ion channel expression and function, Neuron, 14:211–215.

    Article  PubMed  CAS  Google Scholar 

  • McBride, D. W., and Hamill, O. P., 1992, Pressure-clamp: A method for rapid step perturbation of mechanosensitive channels, Pflügers Arch. 412:606–612.

    Article  Google Scholar 

  • McCormack, K., McKormac, T., Tanouye, M., Rudy, B., and Stuhmer, W., 1995, Alternative splicing of the human Shaker K+ channel β1 gene and functional expression of the β2 gene product, FEBS Lett. 370:32–36.

    Article  PubMed  CAS  Google Scholar 

  • McDonald, J. C., Adelman, J. P., Douglass J., and North, A. L., 1989, Expression of a cloned rat brain potassium channel in Xenopus oocytes, Science 24:221–224.

    Google Scholar 

  • 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, Pflügers Arch. 407:577–588.

    Article  PubMed  CAS  Google Scholar 

  • Miledi, R., and Woodward, R. M., 1989, Effects of defolliculation on membrane current responses of Xenopus oocytes, J. Physiol. (London) 416:601–621.

    CAS  Google Scholar 

  • Nagaya, N., and Papazian, D. M., 1997, Potassium channel α and β subunits assemble in the endoplasmic reticulum, J. Biol. Chem. 272:3022–3027.

    Article  PubMed  CAS  Google Scholar 

  • Ohya, Y., and Sperelakis N., 1988, Whole-cell voltage clamp and intracellular perfusion technique on single smooth muscle cells, Mol. Cell. Biochem. 80:79–86.

    PubMed  CAS  Google Scholar 

  • Oron, Y., Gillo, B., and Gershengorn, M. C.,1988, Differences in receptor-evoked membrane electrical responses in native and mRNA-injected Xenopus oocytes, Proc Natl Acad Sci U.S.A. 85:3820–3824.

    Article  PubMed  CAS  Google Scholar 

  • Parekh, A. B., Terlau, H., and Stuhmer, W., 1993, Depletion of InsP3 stores activates a Ca2+ and K+ current by means of a phosphatase and a diffusible messenger, Nature 364:814–818.

    Article  PubMed  CAS  Google Scholar 

  • Penner, R., 1995, A practical guide to patch clamping, in: Single Channel Recording, 2nd ed. (B. Sakmann and E. Neher, eds.). Plenum Press, New York, pp. 3–30.

    Google Scholar 

  • Perozo, E., Papazian, D. M., Stefani, E., and Bezanilla, F., 1992, Gating currents in Shaker K+ channels: Implications for activation and inactivation models, Biophys. J. 62:160–168.

    Article  PubMed  CAS  Google Scholar 

  • Peter, A. B., Schittny, J. C., Niggli, V., Reuter, H., and Segel, E., 1991, The polarized distribution of poly(A+)-mRNA-induced functional ion channels in the Xenopus oocyte plasma membrane is prevented by anticytoskeletal drugs, J. Cell. Biol. 114:455–464.

    Article  PubMed  CAS  Google Scholar 

  • Petersen, K. R., and Nerbonne, J. M., 1999, Expression environment determines K+ current properties: Kv1 and Kv4 alpha-subunit-induced K+ currents in mammalian cell lines and cardiac myocytes, Pflügers Arch. 437:381–392.

    Article  PubMed  CAS  Google Scholar 

  • Potter, H., 1993, Application of electroporation in recombinant DNA technology, Methods Enzymol. 217:461–478.

    Article  PubMed  CAS  Google Scholar 

  • Rae, J., Cooper, K., Gates, P., and Watsky, M., 1991, Low access resistance perforated patch recordings using amphotericin B, J. Neurosci. Methods 37(l):15–26.

    Article  PubMed  CAS  Google Scholar 

  • Rudy, B., 1988, Diversity and ubiquity of K channels, Neuroscience 25:729–749.

    Article  PubMed  CAS  Google Scholar 

  • Rudy, B., Hoger, J. H., Lester, H. A., and Davidson, N., 1988, At least two mRNA species contribute to the properties of rat brain A-type potassium channels expressed in Xenopus oocytes, Neuron 1:649–658.

    Article  PubMed  CAS  Google Scholar 

  • Ruppersberg, J. P., Schroter, K. H., Sakmann, B., Stocker, M., Sewing, S., and Pongs, O., 1990, Heteromultimeric channels formed by rat brain potassium-channel proteins, Nature 345:535–537.

    Article  PubMed  CAS  Google Scholar 

  • Shamotienko, O. G., Parcej, D. N., and Dolly, O., 1997, Subunit combinations defined for K+ channel Kv1 subtypes in synaptic membranes from bovine brain, Biochemistry 36: 8195–8201.

    Article  PubMed  CAS  Google Scholar 

  • Sheng, M., Liao, Y. J., Jan, Y. N., and Jan, L. Y., 1993, Presynaptic A-current based on heteromultimeric K+ channels detected in vivo, Nature 365:72–75.

    Article  PubMed  CAS  Google Scholar 

  • Shi, G., Kleinklaus, A. K., Marrion, N. V., and Trimmer, J. S., 1994, Properties of Kv2.1 K+ channels expressed in transfected mammalian cells, J. Biol. Chem. 269:23204–23211.

    PubMed  CAS  Google Scholar 

  • Shi, G., Nakahira, K., Hammond, S., Rhodes, K. J., Schechter, L. E., and Trimmer, J. S., 1996, β Subunits promote K+ channel surface expression through effects early in biosynthesis, Neuron 16:843–852.

    Article  PubMed  CAS  Google Scholar 

  • Skryma, R., Prevarskaya, N., Vacher, P., and Dufy, B., 1994, Voltage-dependent ionic conductances in Chinese hamster ovary cells, Am. J. Physiol. 267:C544-C553.

    PubMed  CAS  Google Scholar 

  • Sobko, A., Peretz, A., Shirihai, O., Etkin, S., Cherepanova, V., Dagan, D., and Attali, B., 1998, Heteromultimeric delayed-rectifier K+ channels in Schwann cells: Developmental expression and role in cell proliferation, J. Neurosci. 18:10398–10408.

    PubMed  CAS  Google Scholar 

  • Stuhmer, W., 1998, Recordings from Xenopus oocytes, Methods Enzymol. 293:280–300.

    Article  PubMed  CAS  Google Scholar 

  • Stuhmer, W., Methfessel, C., Sakmann, B., Noda, M., and, Numa, S., 1987, Patch clamp characterization of sodium channels expressed from rat brain cDNA, Eur. Biophys. J. 14:131–138.

    Article  PubMed  CAS  Google Scholar 

  • Thornhill, W. B., Wu, M. B., Jiang, X., Wu, X., and Morgan, P. T., 1996, Expression of Kv1.1 delayed rectifier potassium channels in Lec mutant Chinese hamster ovary cell lines reveals a role for sialidation in channel function, J. Biol. Chem. 271:19093–19098.

    Article  PubMed  CAS  Google Scholar 

  • Trimmer, J. S., 1998, Analysis of K+ channel biosynthesis and assembly in transfected mammalian cells, Methods Enzymol. 293:32–49.

    Article  PubMed  CAS  Google Scholar 

  • Trouet, D., Nilius, B., Voets, G. D., and Eggermont, J., 1997, Use of a bicistronic GFP-expression vector to characterize ion channels after transfection in mammalian cells, Pflügers Arch. 434:632–638.

    Article  PubMed  CAS  Google Scholar 

  • Tzounopoulos, T., Maylie, J., and Adelman, J.P., 1995, Induction of endogenous channels by high levels of heterologous membrane proteins in Xenopus oocytes, Biophys. J. 69:904–908.

    Article  PubMed  CAS  Google Scholar 

  • Uebele, V. N., England, S. K., Chaudhary, A., Tamkun, M. M., and Snyders, D. J., 1996, Functional differences in Kv1.5 currents expressed in mammalian cell lines are due to the presence of endogenous Kvβ2.1 subunits, J. Biol. Chem. 271:2406–2412.

    Article  PubMed  CAS  Google Scholar 

  • Wang, Q., and Large, W. A., 1991, Noradrenaline-evoked cation conductance recorded with the nystatin whole-cell method in rabbit portal vein cells, J. Physiol. (London) 435:21–39.

    CAS  Google Scholar 

  • Wang, H., Kunkel, D. D., Martin, T. M., Schwartzkroin, P. A., and Temple, B. L., 1993, Heteromultimeric K+ channels in terminal and juxtaparanodal regions of neurons, Nature 365:75–79.

    Article  PubMed  CAS  Google Scholar 

  • Wischnitzer, S., 1966, The ultrastructure of the cytoplasm of the developing amphibian egg, in: Advances in Morphogenesis, Vol. 5 (M. Abercrombie and J. Brachet, eds.), Academic Press, New York, pp 131–179.

    Google Scholar 

  • Yang, X. C, and Sachs, F., 1989, Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions, Science 243:1068–1071.

    Article  PubMed  CAS  Google Scholar 

  • Yokoshiki H., Katsube Y., Sunugawa M., Seki T., and Sperelakis N., 1997, Disruption of actin cytoskeleton attenuates sulfonylurea inhibition of cardiac ATP-sensitive K+ channels, Pflügers Arch. 434:203–205.

    Article  PubMed  CAS  Google Scholar 

  • Yu, S. P., and Kerchner, G. A., 1998, Endogenous voltage-gated potassium channels in human embryonic kidney (HEK293) cells, J. Neurosci. Res. 52:612–617.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Internal Medicine, Division of Nephrology and Hypertension and Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio, 45267-0576, USA

    Laura Conforti & Nicholas Sperelakis

Authors
  1. Laura Conforti
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nicholas Sperelakis
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Walter McKenzie Health Science Centre, University of Alberta Hospital, Edmonton, Alberta, Canada

    Stephen L. Archer

  2. Medical College of Wisconsin, Milwaukee, Wisconsin, USA

    Nancy J. Rusch

Rights and permissions

Reprints and permissions

Copyright information

© 2001 Springer Science+Business Media New York

About this chapter

Cite this chapter

Conforti, L., Sperelakis, N. (2001). The Patch-Clamp Technique for Measurement of K+Channels in Xenopus Oocytes and Mammalian Expression Systems. In: Archer, S.L., Rusch, N.J. (eds) Potassium Channels in Cardiovascular Biology. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-1303-2_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-4615-1303-2_9

  • Publisher Name: Springer, Boston, MA

  • Print ISBN: 978-1-4613-5487-1

  • Online ISBN: 978-1-4615-1303-2

  • eBook Packages: Springer Book Archive

Publish with us

Policies and ethics

Search

Navigation