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Heterologous expression of the membrane proteins that control cellular excitability

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Part of the book series: EXS ((EXS,volume 63))

Summary

Versatile and potent expression systems are needed to decipher the structure and functions of the many excitability proteins that have been identified through molecular cloning. This article reviews the use of recombinant vaccinia viruses (W), which have been recently explored for the heterologous expression of eukaryotic proteins. Vaccinia viruses feature a series of favourable properties, most of all a broad host range and high efficiency of infection, that make them uniquely suited as flexible expression vectors. In one type of experiment, the recombinant virus simply harbors the cDNA for the foreign protein; in a second type the virus harbors the cDNA for the specific and efficient RNA polymerase of bacteriophage T7, which in turn generates RNA from a separate introduced plasmid or virus. Both variations have been successfully applied to the expression and analysis of voltage-dependent ion channels, neurotransmitter receptors and other excitability proteins in many cell lines and postmitotic cells in culture. W vectors promise to be particularly useful to study membrane proteins that require posttranslational processing, association with cell-specific subunits or coupling to endogenous second messengers pathways.

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References

  • Blakely, R. D., Clark, J. A., Rudnick, G. and Amara, S. G. (1991) Vaceinia-T7 RNA polymerase expression system: evaluation for the expression cloning of plasma membrane transporters. Analyt. Biochem. 194, 302–308.

    Article  Google Scholar 

  • Buller, R. M. L., Smith, G. L., Cremer, K., Notkins, A. L. and Moss, B. (1985) Decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase-negative phenotype. Nature 317, 813–815.

    Article  Google Scholar 

  • Claudio, T., Green, W. N., Hartman, D. S., Hayden, D., Paulson, H. L., Sigworth, F. J., Sine, S. M. and Swedlund, A. (1987) Genetic reconstitution of functional acetylcholine receptor channels in mouse fibroblasts. Science 238, 1688–1694.

    Article  Google Scholar 

  • Claudio, T., Paulson, H. L., Green, W. N., Ross, A. F., Hartman, D. S. and Hayden, D. (1989) Fibroblasts transfected with Torpedo acetylcholine receptor β-, γ-, and δ-subunit cDNAs express functional receptors when infected with a retroviral α recombinant. J. Cell Biol. 108, 2277–2290.

    Article  Google Scholar 

  • Elroy-Stein, O., Fuerst, T. R. and Moss, B. (1989) Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5′ sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system. Proc. Natl Acad. Sci. USA 86, 6126–6130.

    Article  Google Scholar 

  • Franke, C. A. and Hruby, D. E. (1985) Expression of recombinant vaccinia virus derived alphavirus protein in mosquito cells. J. Gen. Virol. 66, 2761–2765.

    Article  Google Scholar 

  • Fuerst, T. R., Niles, E. G., Studier, F. W. and Moss, B. (1986) Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 83, 8122–8126.

    Article  Google Scholar 

  • Fuerst, T. A., Earl, P. L. and Moss, B. (1987) Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes. Molec. Cell. Biol. 7, 2538–2544.

    Google Scholar 

  • George, S. T., Arbabian, M. A., Ruoho, A. E., Kiely, J. and Malbon, C. C. (1989) High-efficiency expression of mammalian β-adrenergic receptors in baculovirus-infected insect cells. Biochem. Biophys. Res. Commun. 163, 1265–1269.

    Article  Google Scholar 

  • Ho, B. Y. Karschin, A., Nolan, C. E., Elroy-Stein, O., Moss, B., Branchek, T., Hartig, P., Lester, H. A. and Davidson, N. (1991) Expression of the serotonin 1A (5-HT1AR) with a vaccinia virus system in mammalian cells. Faseb J. 5, A855 (abstr.).

    Google Scholar 

  • Hsu, H. S., Yang, X. C., Labarca, C., Karschin, A., Elroy-Stein, O., Moss, B., Davidson, N. and Lester, H. A. (1991) Generation of action potentials in previously unexcitable cells. Biophys. J. 59, 41a (abstr.).

    Google Scholar 

  • Jansen, K. U., Conroy, W. G., Claudio, T., Fox, T. D., Fujita, N., Hamill, O., Lindstrom, J. M., Luther, M., Nelson, N., Ryan, K. A., Sweet, M. T. and Hess, G. P. (1989) Expression of the four subunits of the Torpedo californica nicotinic acetylcholine receptor in Saccharomyces cerevisiae. J. Biol. Chem. 264, 1502–1501.

    Google Scholar 

  • Karschin, A., Jayashree-Aiyar, S., Davidson, N. and Lester, H. A. (1990) Vaccinia virus — a unique expression vector for K+ channels in primary cell culture. Eur. J. Neurosci. 3, 257 (abstr.).

    Google Scholar 

  • Karschin, A., Aiyar, J., Gouin, A., Davidson, N. and H. A. Lester (1991a) K+ channel expression in primary cell cultures mediated by vaccinia virus. FEBS Lett. 278, 229–233.

    Article  Google Scholar 

  • Karschin, A., Ho, B. Y., Labarca, C., Elroy-Stein, O., Moss, B., Davidson, N. and Lester, H. A. (1991b) Heterologously expressed serotonin 1A receptors couple to muscarinic K+ channels in heart. Proc. Natl. Acad. Sci. USA 88, 5694–5698.

    Article  Google Scholar 

  • Keown, W. A., Campbell, C. R. and Kucherlapati, R. S. (1990) Methods for introducing DNA into mammalian cells, in: Methods in Enzymology, Vol. 185. Eds J. N. Abelson and M. I. Simon. Academic Press Inc, New York.

    Google Scholar 

  • Kartner, N., Hanrahan, J. W., Jensen, T. J., Naismith, A. L., Sun, S., Ackerley, C. A., Reyes, E. F., Tsui, L.-C., Rommens, J. M., Bear, C. E. and Riordan, J. R. (1991) Expression of the cystic fibrosis gene in non-epithelial invertebrate cells produces a regulated anion conductance. Cell 64, 681–691.

    Article  Google Scholar 

  • Kaufman, R. J. (1990) Vectors used for expression in mammalian cells, in: Methods in Enzymology, Vol. 185. Eds J. N. Abelson and M. I. Simon. Academic Press Inc, New York.

    Google Scholar 

  • Klaiber, K., Williams, N., Roberts, T. M., Papazian, T. M., Jan, L. Y. and Miller, C. (1990) Functional expression of the Shaker K+ channels in a baculovirus-infected insect cell line. Neuron 5, 221–226.

    Article  Google Scholar 

  • King, K., Dohlman, H. G., Thorner, J., Caron, M. G. and Lefkowitz, R. J. (1990) Control of yeast mating signal transduction by a mammalian β2-adrenergic receptor and Gsα subunit. Science 250, 121–123.

    Article  Google Scholar 

  • Leonard, R. J., Karschin, A., Jayashree-Aiyar, S., Davidson, N., Tanouye, M. A., Thomas, L., Thomas, G. and Lester, H. A. (1989) Expression of Drosophila Shaker potassium channels in mammalian cells infected with recombinant vaccinia virus. Proc. Natl. Acad. Sci. USA 86, 7629–7633.

    Article  Google Scholar 

  • Mackett, M., Smith, G. L. and Moss, B. (1982) Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proc. Natl. Acad. Sci. USA 79, 7415–7419.

    Article  Google Scholar 

  • MacKinnon, R., Reinhart, P. H. and White, M. M. (1988) Charybdotoxin block of Shaker K+ channels suggests that different types of K+ channels share common structural features. Neuron 1, 997–1001.

    Article  Google Scholar 

  • Miller, L. K. (1988) Baculoviruses as gene expression vectors. Ann. Rev. Microbiol. 42, 177–199.

    Article  Google Scholar 

  • Moss, B. (1985) Replication of poxviruses, in: Virology. Ed. B. N. Fields. Raven Press, New York.

    Google Scholar 

  • Moss, B. (1990) Regulation of vaccinia virus transcription. Ann. Rev. Biochem. 59, 661–688.

    Article  Google Scholar 

  • Moss, S. J., Smart, T. G., Porter, N. M., Nayeem, N., Devine, J., Stephenson, F. A., MacDonald, R. L. and Barnard, E. A. (1990) Cloned GABA receptors are maintained in a stable cell line: allosteric and channel properties. Eur. J. Pharmacol. 189, 77–88.

    Article  Google Scholar 

  • Nakano, E., Panicali, D. and Paoletti, E. (1982) Molecular genetics of vaccinia virus: demonstration of marker rescue. Proc. Natl Acad. Sci. USA 79, 1593–1596.

    Article  Google Scholar 

  • Pacholczyk, T., Blakely, R. D. and Amara, S. G. (1991) Expression cloning of a cocaine- and antidepressant-sensitive human noradrenaline transporter. Nature 350, 350–354.

    Article  Google Scholar 

  • Panicali, D. and Paoletti, E. (1982) Construction of poxviruses as cloning vectors: Insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccinia virus. Proc. Natl. Acad. Sci. USA 79, 4927–4931.

    Article  Google Scholar 

  • Pritchett, D. B., Sontheimer, H., Gorman, C. M., Kettenmann, H., Seeburg, P. H. and Schofield, P. R. (1988) Transient expression shows ligand gating and allosteric potentiation of GABAA receptor subunits. Science 242, 1306–1308.

    Article  Google Scholar 

  • Rich, D. P., Anderson, M. P., Gregory, R. J., Cheng, S. H., Paul, S., Jefferson, D. M., McCann, J. D., Klinger, K. W., Smith, A. E. and Welsh, J. J. (1990) Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells. Nature 347, 358–363.

    Article  Google Scholar 

  • Shapiro, R. A., Scherer, N. M., Habecker, B. A. Subers, E. M. and Nathanson, N. M. (1988) Isolation, sequence, and functional expression of the mouse Ml muscarinic acetylcho line receptor gene. J. Biol. Chem. 263, 18,397–18, 403.

    Google Scholar 

  • Sigel, E. (1990) Use of Xenopus oocytes for the functional expression of plasma membrane proteins. J. Membr. Biol. 117, 201–221.

    Article  Google Scholar 

  • Smith, G. L. and Moss, B. (1983) Infectious poxvirus vectors have capacity for at least 25,000 bp of foreign DNA. Gene 25, 21–28.

    Article  Google Scholar 

  • Talib, S., Leiby, K. R., Wright, K. and Okarma, T. B. (1991) Cloning and expression in Escherichia coli of a synthetic gene encoding the extracellular domain of the human muscle acetylcholine receptor a-subunit. Gene 98, 289–293.

    Article  Google Scholar 

  • Tanabe, T., Beam, K. G., Powell, J. A. and Numa, S. (1988) Restoration of excitation-contraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA. Nature 336, 134–139.

    Article  Google Scholar 

  • Vasudevan, S., Reiländer, H., Maul, G. and Michel H. (1991) Expression and cell membrane localization of rat M3 muscarinic acetylcholine receptor produced in Sf9 insect cells using the baculo virus system. FEBS Lett. 283, 52–56.

    Article  Google Scholar 

  • Whiting, P., McKernan, R. M. and Iversen, L. L. (1990) Another mechanism for creating diversity in γ-aminobutyric type A receptors: RNA splicing directs expression of two forms of γ2 subunit, one of which contains a protein kinase C phosphorylation site. Proc. Natl. Acad. Sci. USA 87, 9966–9970.

    Article  Google Scholar 

  • Yang, X.-C., Karschin, A., Labarca, C., Elroy-Stein, O., Moss, B., Davidson, N. and Lester, H. A. (1991) Expression of ion channels and receptors in Xenopus oocytes using vaccinia virus. Faseb J. 5, 2209–2216.

    Google Scholar 

  • Yang, X.-C., Labarca, C., Nargeot, J., Ho, B. Y., Elroy-Stein, O., Moss, B., Davidson, N. and H. A. Lester (1992) Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel α subunit expressed in mammalian cells. J. Neurosci., in the press.

    Google Scholar 

  • Zagotta, W. N., Germeraad, S., Garber, S. S., Hoshi, T. and Aldrich, R. W. (1989) Properties of the ShB A-type potassium channels expressed in Shaker mutant Drosophila by germline transformation. Neuron 3, 773–782.

    Article  Google Scholar 

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© 1993 Birkhäuser Verlag Basel/Switzerland

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Karschin, A. (1993). Heterologous expression of the membrane proteins that control cellular excitability. In: Pichon, Y. (eds) Comparative Molecular Neurobiology. EXS, vol 63. Birkhäuser Basel. https://doi.org/10.1007/978-3-0348-7265-2_2

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  • DOI: https://doi.org/10.1007/978-3-0348-7265-2_2

  • Publisher Name: Birkhäuser Basel

  • Print ISBN: 978-3-0348-7267-6

  • Online ISBN: 978-3-0348-7265-2

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