Differential effects of mutations on the transport properties of the Na+/H+ antiporter NhaA from Escherichia coli

Thomas Mager, Markus Braner, Bastian Kubsch, Lina Hatahet, Dudu Alkoby, Abraham Rimon, Etana Padan, Klaus Fendler*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

Background: NhaA of Escherichia coli can serve as an example for pH regulation and transport mechanism in Na+/H+ exchangers. Results: Different mutations alter Na+ and/or H+ binding or conformational dynamics. Conclusion: Mutations in different segments of the protein change its pH profile by affecting different aspects of its transport mechanism. Significance: Single site mutations and kinetic analysis reveal determinants of Na+/H+ exchanger mechanism. 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.

Original languageEnglish
Pages (from-to)24666-24675
Number of pages10
JournalJournal of Biological Chemistry
Volume288
Issue number34
DOIs
StatePublished - 23 Aug 2013

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