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Tagging Ion Channels with the Green Fluorescent Protein (GFP) as a Method for Studying Ion Channel Function in Transgenic Mouse Models

  • Joseph C. Koster
  • Colin G. Nichols
Part of the Methods in Pharmacology and Toxicology book series (MIPT)

Abstract

In the past ten years, a multitude of cDNAs encoding ion channels have been cloned and have led to a greater understanding of the biological aspects of ion-channel function. Relevant information on structure, function, and pharmacology has come primarily from expressing reconstituted wild-type or mutant ion channels in heterologous expression systems and assaying channel activity using standard biochemical and electrophysiological techniques. In particular, Xenopus oocytes have proven to be a useful expression system owing to the high level of expression of functional ion channels at the cell surface and the large size of the oocyte, which makes it particularly amenable to ion channel analysis using various electrophysiological techniques. However, interpretation of the functional data can be complicated by the presence of several endogenous oocyte currents that can be induced upon exogenous channel expression (1,2). Expressing cloned ion channels in cultured mammalian cells using the transfection technique can often overcome the problem of superimposed endogenous currents while providing an appropriate model of the mammalian cell. This is especially important when addressing questions of channel protein-processing, pharmacology, and roles of ion channels in signal transduction. Ultimately, however, questions of ion-channel physiology and a potential role of altered channel activity in the disease state requires expression in the whole animal model. Numerous ion channels have now been expressed in a tissue-specific fashion in genetically defined strains of mice using the transgenic expression system (3, 4, 5, 6, 7, 8). With a rise in the number of core facilities at most research institutions to generate and house transgenic mouse colonies, as well the increased affordability, the transgenic mouse model has become an increasingly important and informative tool for many researchers. In brief, a linearized DNA construct containing the ion-channel cDNA under control of a tissuespecific promoter, the “transgene,” is microinjected into the pronuclei of mouse zygotes. Generally, the transgene integrates into the mouse chromosomal DNA at the one cell-stage, so it is present in every cell of the animal and is capable of being transmitted through the germ line of the adult mouse. Although integration of the transgene usually occurs at a single site in the genome, the site itself is usually random and may contain several copies of the transgene. Theoretically, each founder mouse may express the transgene differently based on the number of transgene copies present and the site of the integration event (however, copy number in itself is not an adequate predictor of the level of transgene expression [9]). Moreover, analysis of the transgene can be further complicated owing to intrafamily variation of expression (10) within a founder line as well as variegated expression within the target tissue itself (11) (i.e., not every cell within the target tissue expresses the transgene or expression levels vary from cell to cell despite the fact that every cell contains the transgene, see Figs. 2 and Figs. 3). For these reasons, determining the level of transgene expression is critical when comparing phenotypes of different transgenic mouse lines and analyzing the physiology of individual cells within the target tissue. In this regard, we now describe a detection method for transgenic mice that uses variant of the green fluorescent protein (GFP) to tag a transgenically expressed ion channel. As demonstrated below, addition of the GFP-tag has no deleterious effect on channel activity and allows for easy and efficient detection of the GFP-tagged ion channels in individual cells of the transgenic animal using UV illumination. Moreover, as fluorescence intensity is related to the level of transgene expression, this method allows for a quick and qualitative assessment of transgene expression on a cell to cell basis.

Keywords

Green Fluorescent Protein KATP Channel Founder Line Mouse Zygote Transgenic Expression System 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Weber, W. M. (1999) Endogenous ion channels in oocytes of Xenopus laevis: recent developments. J. Membrane Biol. 170, 1–12.CrossRefGoogle Scholar
  2. 2.
    Attali, B., Guillemare, E., Lesage, F., Honoré, E., Romey, G., Lazdunski, M., and Barhanin, J. (1993) The protein IsK is a dual activator of K+ and Cl channels. Nature 365, 850–852.PubMedCrossRefGoogle Scholar
  3. 3.
    Sutherland, M. L., Williams, S. H., Abedi, R., Overbeek, P. A., Pfaffinger, P. J., and Noebels, J. L. (1999) Overexpression of a Shaker-type potassium channel in mammalian central nervous system dysregulates native potassium channel gene. Proc. Natl. Acad. Sci. USA 96, 2451–2455.PubMedCrossRefGoogle Scholar
  4. 4.
    Xu, H. and Nerbonne, J. M. (1999) Elimination of the transient outward current and action potential prolongation in mouse atrial myocytes expressing a dominant negative Kv4 alpha subunit. J. Physiol. 519, 11–21.PubMedCrossRefGoogle Scholar
  5. 5.
    Philipson, L. H., Rosenberg, M. P., Kuznetsov, A., Lancaster, M. E., Worley, J. F. III, Roe, M. W., and Dukes, I. D. (1994) Delayed rectifier K+ channel overexpression in transgenic islets and β-cells associated with impaired glucose responsiveness. J. Biol. Chem. 269, 27,787–27,790.Google Scholar
  6. 6.
    Miki, T., Tashiro, F., Iwanaga, T., Nagashima, K., Yoshitomi, H., Aihara, H., et al. (1997) Abnormalities of pancreatic islets by targeted expression of a dominant-negative KATP channel. Proc. Natl. Acad. Sci. USA 91, 11,969–11,973.CrossRefGoogle Scholar
  7. 7.
    Babij, P., Askew, G. R., Nieuwenhuijsen, B., Su, C. M., Bridal, T. R., Jow, B., et al. (1998) Inhibition of cardiac delayed rectifier K+-current by overexpression of the long-QT syndrome HERG G628S mutation in transgenic mice. Circ. Res. 83, 668–678.PubMedGoogle Scholar
  8. 8.
    Muth J. N., Yamaguchi, H., Mikala, G., Grupp, I. L., Lewis, W., Cheng, H., et al. (1999) Cardiac-specific overexpression of the alpha(1) subunit of the Ltype voltage-dependent Ca(2+) channel in transgenic mice. Loss of isoproterenol-induced contraction. J. Biol. Chem. 274, 21,503–21,506.PubMedCrossRefGoogle Scholar
  9. 9.
    Bieberich, C. J., Utset, M. F., Awgulewitsch, A., and Ruddle, F. H. (1990) Evidence for positive and negative regulation of the Hox-3. 1 gene. Proc. Natl. Acad. Sci. USA 87, 8462–8466.PubMedCrossRefGoogle Scholar
  10. 10.
    Overbeek, P. (1994) Factors affecting transgenic animal production, in Transgenic Animal Technology: A Laboratory Handbook (Pinkert, C. A., ed.), Academic, New York, pp. 96–107.Google Scholar
  11. 11.
    Dobie, K. W., Lee, M., Fantes, J. A., Graham, E., Clark, A. J., Springbett, A., et al. (1996) Variegated transgene expression in mouse mammary gland is determined by the transgenic integration locus. Proc. Natl. Acad. Sci. USA 93, 6659–6664.PubMedCrossRefGoogle Scholar
  12. 12.
    Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., and Cormier, M. J. (1992) Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111, 229–233.PubMedCrossRefGoogle Scholar
  13. 13.
    Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., Prasher, D. C. (1994) Green fluorescent protein as a marker for gene expression. Science 263(5148), 802–805.PubMedCrossRefGoogle Scholar
  14. 14.
    Patterson, G. H., Knobel, S. M., Sharif, W. D., Kain, S. R., and Piston, D. W. (1997) Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys. J. 73, 2782–2790.PubMedCrossRefGoogle Scholar
  15. 15.
    Ashcroft, F. M. (1988) Adenosine 5’-triphosphate-sensitive potassium channels. Annu. Rev. Neurosci. 11, 97–118.PubMedCrossRefGoogle Scholar
  16. 16.
    Inagaki, N., Gonoi, T., Clement, J. P. IV, Namba, N., Inazawa, J., Gonzales, G., et al. (1995) Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor. Science 270, 1166–1170.PubMedCrossRefGoogle Scholar
  17. 17.
    Inagaki, N., Gonoi, T., and Seino, S. (1997) Subunit stoichiometry of the pancreatic b-cell ATP-sensitive K+ channel. FEBS Lett. 409, 232–236.PubMedCrossRefGoogle Scholar
  18. 18.
    Clement, J. P. IV, Kunjilwar, K., Gonzalez, G., Schwanstecher, M., Panten, U., Aguilar Bryan, L., and Bryan, J. (1997) Association and stoichiometry of KATP channel subunits. Neuron 18, 827–838.PubMedCrossRefGoogle Scholar
  19. 19.
    Koster, J. C., Sha, Q., Shyng, S.-L., and Nichols, C. G. (1999a) ATP Inhibition of KATP Channels: Control of nucleotide sensitivity by the N-terminal domain of the Kir6. 2 subunit. J. Physiol. 515, 19–30.PubMedCrossRefGoogle Scholar
  20. 20.
    Kupper, J. (1998) Functional expression of GFP-tagged Kv1. 3 and Kv1. 4 channels in HEK 293 cells. Eur. J. Neurosci. 10, 3908–3912.PubMedCrossRefGoogle Scholar
  21. 21.
    Bueno, O. F., Robinson, L. C., Alvarez-Hernandez, X., and Leidenheimer, N. J. (1998) Functional characterization and visualization of a GABAA receptor-GFP chimera expressed in Xenopus oocytes. Brain Res. Mol. Brain Res. 59, 165–177.PubMedCrossRefGoogle Scholar
  22. 22.
    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. 510, 333–345.PubMedCrossRefGoogle Scholar
  23. 23.
    Moyer, B. D., Loffing, J., Schwiebert, E. M., Loffing-Cueni, D., Halpin, P. A., Karlson, K. H., et al. (1998) Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator, tagged with green fluorescent protein in madin-darby canine kidney cells. J. Biol. Chem. 273, 21,759–21,768.PubMedCrossRefGoogle Scholar
  24. 24.
    Grabner, M., Dirksen, R. T., and Beam, K. G. (1998) Tagging with green fluorescent protein reveals a distinct subcellular distribution of L-type and non-Ltype Ca2+ channels expressed in dysgenic myotubes. Proc. Natl. Acad. Sci. USA 95, 1903–1908.PubMedCrossRefGoogle Scholar
  25. 25.
    Makhina, E. N. and Nichols, C. G. (1998) Independent trafficking of KATP channel subunits to the plasma membrane. J. Biol. Chem. 273, 3369–3374.PubMedCrossRefGoogle Scholar
  26. 26.
    Meyer, E. and Fromherz, P. (1999) Ca2+ activation of hSlo K+ channel is suppressed by N-terminal GFP tag. Euro. J. Neurosci. 11, 1105–1108.CrossRefGoogle Scholar
  27. 27.
    Kisseberth, W. C., Brettingen, N. T., Lohse, J. K., and Sandgren, E. P. (1999) Ubiquitous expression of marker transgenes in mice and rats. Develop. Biol. 214, 128–138.PubMedCrossRefGoogle Scholar
  28. 28.
    Palermo, J., Gulick, J., Ng, W., Grupp, I. L., Grupp, G., and Robbins J. (1995) Remodeling the mammalian heart using Tran genesis. Cell. Mol. Biol. Res. 41, 501–509.PubMedGoogle Scholar
  29. 29.
    Dandoy-Dron, F., Monthioux, E., Jami, J., and Bucchini, D. (1991) Regulatory regions of rat insulin I gene necessary for expression in transgenic mice. Nucleic Acids Res. 19, 4925–4930.PubMedCrossRefGoogle Scholar
  30. 30.
    Sakura, H., Ashcroft, S. J., Terauchi, Y., Kadowaki, T., and Ashcroft, F. M. (1998) Glucose modulation of ATP-sensitive K-currents in wild-type, homozygous and heterozygous glucokinase knock-out mice. Diabetologia 41, 654–659.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2001

Authors and Affiliations

  • Joseph C. Koster
    • 1
  • Colin G. Nichols
    • 1
  1. 1.Department of Cell Biology and PhysiologyWashington University Medical SchoolSt. Louis

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