title: Functional complementation and genetic deletion studies of KirBac channels: activatory mutations highlight gating-sensitive domains.
abstract: The superfamily of prokaryotic inwardly rectifying (KirBac) potassium channels is homologous to mammalian Kir channels. However, relatively little is known about their regulation or about their physiological role in vivo. In this study, we have used random mutagenesis and genetic complementation in K(+)-auxotrophic Escherichia coli and Saccharomyces cerevisiae to identify activatory mutations in a range of different KirBac channels. We also show that the KirBac6.1 gene (slr5078) is necessary for normal growth of the cyanobacterium Synechocystis PCC6803. Functional analysis and molecular dynamics simulations of selected activatory mutations identified regions within the slide helix, transmembrane helices, and C terminus that function as important regulators of KirBac channel activity, as well as a region close to the selectivity filter of KirBac3.1 that may have an effect on gating. In particular, the mutations identified in TM2 favor a model of KirBac channel gating in which opening of the pore at the helix-bundle crossing plays a far more important role than has recently been proposed.
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PU, United Kingdom.
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Paynter
authors.forename: Isabelle
authors.initials: I
authors.lastname: Andres-Enguix
authors.forename: Philip W
authors.initials: PW
authors.lastname: Fowler
authors.forename: Stephen
authors.initials: S
authors.lastname: Tottey
authors.forename: Wayland
authors.initials: W
authors.lastname: Cheng
authors.forename: Decha
authors.initials: D
authors.lastname: Enkvetchakul
authors.forename: Vassiliy N
authors.initials: VN
authors.lastname: Bavro
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Kusakabe
authors.forename: Mark S P
authors.initials: MS
authors.lastname: Sansom
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Robinson
authors.forename: Colin G
authors.initials: CG
authors.lastname: Nichols
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Tucker
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D015640:Ion Channel Gating / Q000502:physiology; D009154:Mutation; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D046939:Synechocystis / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D024661:Potassium Channels, Inwardly Rectifying; D029701:Saccharomyces cerevisiae Proteins