title: Genetic selection of activatory mutations in KcsA.
abstract: The KcsA potassium channel from Streptomyces lividans is one of the most actively studied ion channels. However, there are still unresolved issues about its gating mechanism in vivo because the channel is only activated by highly acidic intracellular pH, meaning that it will be mostly inactive in its host environment. In this study we have used a genetic complementation assay of K+-auxotrophic E. coli (TK2420) and S. cerevisiae (SGY1528) to identify activatory or 'gain-of-function' mutations which allow functional activity of KcsA in the physiological environment of two markedly different expression systems. These mutations clustered at the helix-bundle-crossing in both TM1 and TM2 (residues H25, L105, A108, T112, W113, F114, E118 and Q119), and include residues previously implicated in the pH-gating mechanism. We discuss how these gain-of-function mutations may result in their activatory phenotype, the relative merits of the E. coli and S. cerevisiae genetic complementation approaches for the identification of gating mutations in prokaryotic K+ channels, and ways in which this assay may be improved for future use in screening protocols.
authors.affiliation: Oxford Centre for Gene Function, Department of Physiology Anatomy and Genetics, University of Oxford, Oxford, UK.
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Paynter
authors.forename: Peter
authors.initials: P
authors.lastname: Sarkies
authors.forename: Isabelle
authors.initials: I
authors.lastname: Andres-Enguix
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Tucker
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D009154:Mutation / Q000502:physiology*; D015221:Potassium Channels / Q000235:genetics*; D024642:Potassium Channels, Voltage-Gated; D048170:Streptomyces lividans
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C504448:KcsA protein, Streptomyces coelicolor; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; C107790:prokaryotic potassium channel
references.citation: Biochemistry. 2003 Aug 12;42(31):9263-8
references.pmid: 12899612
references.citation: Biophys J. 2004 Sep;87(3):1526-36
references.pmid: 15345533
references.citation: Biochem Soc Trans. 2005 Nov;33(Pt 5):916-20
references.pmid: 16246010
references.citation: Biochemistry. 2002 Nov 19;41(46):13653-62
references.pmid: 12427027
references.citation: J Gen Physiol. 2001 Feb;117(2):165-80
references.pmid: 11158168
references.citation: EMBO Rep. 2006 Jun;7(6):611-6
references.pmid: 16641935
references.citation: Biochem Biophys Res Commun. 2004 Apr 2;316(2):429-36
references.pmid: 15020236
references.citation: Cell. 1999 Mar 19;96(6):879-91
references.pmid: 10102275
references.citation: Microbiology (Reading). 2000 Oct;146 ( Pt 10):2671-2678
references.pmid: 11021942
references.citation: Neuron. 2005 Sep 15;47(6):833-43
references.pmid: 16157278
references.citation: Microbiology (Reading). 2006 Sep;152(Pt 9):2831-2841
references.pmid: 16946277
references.citation: Nat Neurosci. 2003 Apr;6(4):353-61
references.pmid: 12640457
references.citation: Proc Natl Acad Sci U S A. 2004 Mar 30;101(13):4441-6
references.pmid: 15070737
references.citation: Nucleic Acids Res. 1987 Apr 24;15(8):3581-93
references.pmid: 3554144
references.citation: Proc Natl Acad Sci U S A. 2007 Mar 13;104(11):4342-6
references.pmid: 17360526
references.citation: Nat Struct Mol Biol. 2006 Apr;13(4):311-8
references.pmid: 16532009
references.citation: Neuron. 2001 Mar;29(3):657-67
references.pmid: 11301025
references.citation: Proc Natl Acad Sci U S A. 2008 May 13;105(19):6900-5
references.pmid: 18443286
references.citation: Science. 1999 Jul 2;285(5424):73-8
references.pmid: 10390363
references.citation: Biochim Biophys Acta. 2007 May;1768(5):1218-29
references.pmid: 17336921
references.citation: J Bacteriol. 1988 Oct;170(10):4727-31
references.pmid: 2844726
references.citation: Prog Nucleic Acid Res Mol Biol. 2003;75:293-320
references.pmid: 14604015
references.citation: Nat Struct Mol Biol. 2008 Jun;15(6):605-12
references.pmid: 18488040
references.citation: J Biol Chem. 2006 Jul 28;281(30):21131-21138
references.pmid: 16728395
references.citation: Nat Struct Mol Biol. 2006 Apr;13(4):319-22
references.pmid: 16532008
references.citation: Channels (Austin). 2007 Sep-Oct;1(5):327-30
references.pmid: 18690035
references.citation: J Biotechnol. 1995 Jan 15;38(2):165-72
references.pmid: 7765807
references.citation: Neuron. 2007 Aug 16;55(4):602-14
references.pmid: 17698013
references.citation: Mol Pharmacol. 2004 Jan;65(1):214-9
references.pmid: 14722253
references.citation: FEBS Lett. 1982 Mar 8;139(1):121-4
references.pmid: 7042384
references.citation: Nature. 2001 Nov 1;414(6859):43-8
references.pmid: 11689936
references.citation: J Biol Chem. 2007 May 18;282(20):15179-86
references.pmid: 17360718
references.citation: Proc Natl Acad Sci U S A. 2005 Dec 6;102(49):17630-5
references.pmid: 16301524
references.citation: Methods Mol Biol. 2006;337:157-65
references.pmid: 16929946
references.citation: Physiol Genomics. 2006 Jun 16;26(1):1-7
references.pmid: 16595742