Abstract
Transient transfectron of mammalian cell lines with recombinant DNA has become a common tool for studying functional and structural properties of a wide variety of proteins (1–5). Transient transfections are especially practical for the expression of recombinant multisubunit complexes because their expression needs the prior knowledge and selection of which of a set of subunits are necessary to form functional heteromultrmers with distinct features. Subumt stoichiometry, mode of assembly, and the relative expression levels of individual cDNA vector constructs also have to be taken into account. The latter still poses an insurmountable problem, because no accurate predictions can be made.
Keywords
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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Keinären K. Wisden W. Sommer B. Werner P. Herb A. Verdoorn T. A. Sakmann B., and Seeburg P. H. (1990) A family of AMPA-selective glutamate receptors. Science 249, 556–560.
Lüddens H. Pritchett D. B. Köhler M. Killisch I. Keinànen K. Monyer H. Sprengel R., and Seeburg P. H. (1990) Cerebellar GABAA receptor selective for a behavioural alcohol antagonist Nature 346, 648–651.
Matthew P. A. Mason J. I. Trant J. M., and Waterman M. R. (1990) Incorporation of steroidogenic pathways which produce cortisol and aldosterone from cholesterol into nonsteroidogenic cells. Mol. Cell Endocrinol. 73, 73–80.
Wei L. Alhenc G. F. Soubrier F. Michaud A. Corvol P., and Clauser E. (1991) Expression and characterization of recombinant human angiotensin I-converting enzyme: evidence for a C-termmal transmembrane anchor and for a proteolytic processing of the secreted recombinant and plasma enzymes. J. Bzol. Chem. 266, 5540–5546.
Nakai A. Sakurai A. Macchia E Fang V., and DeGroot L. J (1990) The roles of three forms of human thyroid hormone receptor in gene regulation. Mol Cell Endocrinol 72, 143–148.
Chen C. and Okayama H. (1987) High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell Blol. 7, 2745–2752.
Behr J. P. Demeneix B. Loeffler J. P. Perez and Mutul J. (1989) Efficient gene transfer into mammalian pnmary endocrine cells with lipopolyamine-coated DNA. Proc Natl. Acad SCL USA 86, 6982–6986.
Felgner P. L. Gadek T. R. Holm M. Roman R. Chan H. W. Wenz M. Northrop J. P. Ringold G. M., and Danielsen M. (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl Acad. Sci. USA 84, 7413–7417.
Felgner J. H. Kumar R. Sridhar C. N. Wheeler C J. Tsai Y. J. Border R. Ramsey P. Martin M., and Felgner P. L. (1994) Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J Biol. Chem. 269, 2550–2561.
Neumann E. Schaefer Ridder M. Wang Y, and Hofschneider P. H (1982) Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBOJ 1, 841–845.
Johnston S. A. (1990) Biolistic transformation: microbes to mice. Nature 346, 776, 777.
Cutting G. R. Lu L. Ohara B. F. Kasch L. M. Montrose-Rafizadeh C. Donovan D. M. Shimada S. Antonarakis S. E. Guggino W. B. Uhl G. R., and Kazazian H. H. (1991) Cloning of the γ-aminobutyric acid (GABA) p1 cDNA: a GABA receptor subunit highly expressed in the retina. Proc. Natl. Acad, Sci. USA 88, 2673–2677.
Seeburg P. H. Wisden W. Verdoorn T. A. Pritchett D. B. Werner P. Herb A. Lüddens H. Sprengel R., and Sakmann B. (1990) The GABAA receptor family: molecular and functional diversity. Coldspring Harbour Symp. Quant. Biol 55, 29–44.
Lùddens H. and Wisden W. (1991) Functron and pharmacology of multiple GABAA receptor subunits, Trends Pharmacol. Sci. 12, 49–51.
Sieghart W. (1995) Structure and pharmacology of γ-aminobutyric acidA receptor subtypes. Pharmacol. Rev. 47, 181–234.
Schofield P. R. Darlison M. G. Fujita N. Burt D. R. Stephenson F. A. Rodriguez H. Rhee L. M. Ramachandran J. Reale V. Glencorse T. A. Reale V. Seeburg P. H., and Barnard E. A. (1987) Sequence and functional expression of the GABAA receptor shows a ligand-gated receptor super-family. Nature 328, 22l–227.
Levitan E. S. Schofield P. R. Burt D. R. Rhee L. M. Wisden W. Kohler M. Fujita N. Rodriguez H. F. Stephenson F. A. Darlison M. G. Barnard E., and Seeburg P. H. (1988) Structural and functional basis for GABAA receptor heterogeneity. Nature 335, 76–79
Pritchett D. B. Sontheimer H. Shivers B. D. Ymer S. Kettenmann H. Schofield P. R., and Seeburg P. H. (1989) Importance of a novel GABAA receptor subunit for benzodrazepine pharmacology. Nature 338, 582–585.
Sigel E. Baur R. Trube G. Möhler H., and Malherbe P. (1990) The effect of subunit composition of rat brain GABAA receptors on channel function. Neuron 5, 703–711.
Perez Velazquez J L. and Angelides K. J. (1993) Assembly of GABAA receptor subumts determines sorting and localization in polarized cells. Nature 361, 457–460.
MacDonald R. L. and Olsen R. W. (1994) GABAA receptor channels. Annu Rev. Neurosci. 17, 569–602.
Shivers B. D. Killisch I. Sprengel R. Sontheimer H. Kohler M. Schofield P. R., and Seeburg P. H. (1989) Two novel GABAA receptor subumts exist in distinct neuronal subpopulations. Neuron 3, 327–337
Moody E. J. Suzdak P. D. Paul S. M., and Skolnick P. (1988) Modulation of the benzodiazepine/γ-aminobutyric acid receptor chloride channel complex by inhalation anesthetics. J. Neurochem. 51, 1386–1393.
Korpi E. R. and Lüddens H. (1993) Regional γ-aminobutyric acid sensitivity of t-butyl-bicyclophosphoro[35S]thionate binding depends on γ-ammobutyric acidA-receptor a subunit. Mol. Pharmacol 44, 87–92.
Lüddens H. Seeburg P. H. S. and Korpi E. R. (1994) Impact of β and γ variants on ligand binding properties of γ-aminobutyric acid type A receptors. Mol. Pharmacol. 45, 810–814.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1997 Humana Press Inc.
About this protocol
Cite this protocol
Lüddens, H., Korpi, E.R. (1997). Methods for Transient Expression of Hetero-Oligomeric Ligand-Gated Ion Channels. In: Challiss, R.A.J. (eds) Receptor Signal Transduction Protocols. Methods in Molecular Biology™, vol 83. Humana Press. https://doi.org/10.1385/0-89603-495-X:55
Download citation
DOI: https://doi.org/10.1385/0-89603-495-X:55
Publisher Name: Humana Press
Print ISBN: 978-0-89603-495-2
Online ISBN: 978-1-59259-567-9
eBook Packages: Springer Protocols