title: Random mutagenesis screening indicates the absence of a separate H(+)-sensor in the pH-sensitive Kir channels.
abstract: Several inwardly-rectifying (Kir) potassium channels (Kir1.1, Kir4.1 and Kir4.2) are characterised by their sensitivity to inhibition by intracellular H(+) within the physiological range. The mechanism by which these channels are regulated by intracellular pH has been the subject of intense scrutiny for over a decade, yet the molecular identity of the titratable pH-sensor remains elusive. In this study we have taken advantage of the acidic intracellular environment of S. cerevisiae and used a K(+) -auxotrophic strain to screen for mutants of Kir1.1 with impaired pH-sensitivity. In addition to the previously identified K80M mutation, this unbiased screening approach identified a novel mutation (S172T) in the second transmembrane domain (TM2) that also produces a marked reduction in pH-sensitivity through destabilization of the closed-state. However, despite this extensive mutagenic approach, no mutations could be identified which removed channel pH-sensitivity or which were likely to act as a separate H(+) -sensor unique to the pH-sensitive Kir channels. In order to explain these results we propose a model in which the pH-sensing mechanism is part of an intrinsic gating mechanism common to all Kir channels, not just the pH-sensitive Kir channels. In this model, mutations which disrupt this pH-sensor would result in an increase, not reduction, in pH-sensitivity. This has major implications for any future studies of Kir channel pH-sensitivity and explains why formal identification of these pH-sensing residues still represents a major challenge.
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, UK.
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Paynter
authors.forename: Lijun
authors.initials: L
authors.lastname: Shang
authors.forename: Murali K
authors.initials: MK
authors.lastname: Bollepalli
authors.forename: Thomas
authors.initials: T
authors.lastname: Baukrowitz
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Tucker
mesh_terms: D019943:Amino Acid Substitution; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D016296:Mutagenesis; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae
chemical_list: D024661:Potassium Channels, Inwardly Rectifying