title: Differential effects of mutations on the transport properties of the Na+/H+ antiporter NhaA from Escherichia coli.
abstract: Na(+)/H(+) antiporters show a marked pH dependence, which is important for their physiological function in eukaryotic and prokaryotic cells. In NhaA, the Escherichia coli Na(+)/H(+) antiporter, specific single site mutations modulating the pH profile of the transporter have been described in the past. To clarify the mechanism by which these mutations influence the pH dependence of NhaA, the substrate dependence of the kinetics of selected NhaA variants was electrophysiologically investigated and analyzed with a kinetic model. It is shown that the mutations affect NhaA activity in quite different ways by changing the properties of the binding site or the dynamics of the transporter. In the first case, pK and/or KD(Na) are altered, and in the second case, the rate constants of the conformational transition between the inside and the outside open conformation are modified. It is shown that residues as far apart as 15-20 Å from the binding site can have a significant impact on the dynamics of the conformational transitions or on the binding properties of NhaA. The implications of these results for the pH regulation mechanism of NhaA are discussed.
authors.affiliation: Max-Planck-Institut für Biophysik, 60438 Frankfurt/Main, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Mager
authors.forename: Markus
authors.initials: M
authors.lastname: Braner
authors.forename: Bastian
authors.initials: B
authors.lastname: Kubsch
authors.forename: Lina
authors.initials: L
authors.lastname: Hatahet
authors.forename: Dudu
authors.initials: D
authors.lastname: Alkoby
authors.forename: Abraham
authors.initials: A
authors.lastname: Rimon
authors.forename: Etana
authors.initials: E
authors.lastname: Padan
authors.forename: Klaus
authors.initials: K
authors.lastname: Fendler
mesh_terms: D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport / Q000502:physiology; D008954:Models, Biological; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; C102839:NhaA protein, E coli; D017923:Sodium-Hydrogen Exchangers
keywords: Electrophysiology; Membrane Transport; Site-directed Mutagenesis; Sodium-Proton Exchange; Transport Mechanism; pH Regulation