Abstract
Potassium channels play an essential role in a wide range of biological processes, including cell volume regulation and the maintenance and control of electrical signals. With the advent of the structural era of ion channel biology, it has become critical to learn more about the functional properties of the prokaryotic channels, and this is the area in which genetic screens have become an increasingly useful approach. Here, we describe a bacteria-based complementation assay that we applied to investigate gating mutants of the prokaryotic K+ channel MthK, which was cloned from the archeon Methanobacterium thermoautotrophicum. The results demonstrated that heterologously expressed MthK is fully assembled and functional in Escherichia coli. This complementation assay should be useful in the initial identification of prokaryotic K+ channel mutants that result in altered channel function.
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References
Doyle, D. A., Morais Cabral, J., Pfuetzner, R. A., et al. (1998) The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280, 69–77.
Bertl, A., Anderson, J. A., Slayman, C. L., Sentenac, H., and Gaber, R. F. (1994) Inward and outward rectifying potassium currents in Saccharomyces cerevisiae mediated by endogenous and heterologously expressed ion channels. Folia Microbiol. (Praha) 39, 507–509.
Yi, B. A., Lin, Y. F., Jan, Y. N., and Jan, L. Y. (2001) Yeast screen for constitutively active mutant G protein-activated potassium channels. Neuron 29, 657–667.
Irizarry, S. N, Kutluay, E., Drews, G., Hart, S. J., and Heginbotham, L. (2002) Opening the KcsA K+ channel: tryptophan scanning and complementation analysis lead to mutants with altered gating. Biochemistry 41, 13,653–13,662.
Hellmer, J., and Zeilinger, C. (2003) MjK1, a K+ channel from M. jannaschii, mediates K+ uptake and K+ sensitivity in E. coli. FEBS Lett. 547, 165–169.
Jiang, Y., Lee, A., Chen, J., Cadene, M., Chait, B. T., and MacKinnon, R. (2002) Crystal structure and mechanism of a calcium-gated potassium channel. Nature 417, 515–522.
Schlösser, A., Meldorf, M., Stumpe, S., Bakker, E. P., and Epstein, W. (1995) TrkH and its homolog, TrkG, determine the specificity and kinetics of cation transport by the Trk system of Escherichia coli. J. Bacteriol. 177, 1908–1910.
Bossemeyer, D., Borchard, A, Dosch, D. C., et al. (1989) K+-transport protein TrkA of Escherichia coli is a peripheral membrane protein that requires other trk gene products for attachment to the cytoplasmic membrane. J. Biol. Chem. 264, 16,403–16,410.
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© 2006 Humana Press Inc.
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Parfenova, L.V., Rothberg, B.S. (2006). Genetic Screening for Functionality of Bacterial Potassium Channel Mutants Using K+ Uptake-Deficient Escherichia coli . In: Stockand, J.D., Shapiro, M.S. (eds) Ion Channels. Methods in Molecular Biology™, vol 337. Humana Press. https://doi.org/10.1385/1-59745-095-2:157
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DOI: https://doi.org/10.1385/1-59745-095-2:157
Publisher Name: Humana Press
Print ISBN: 978-1-58829-576-7
Online ISBN: 978-1-59745-095-9
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