TY - JOUR
T1 - Differential effects of mutations on the transport properties of the Na+/H+ antiporter NhaA from Escherichia coli
AU - Mager, Thomas
AU - Braner, Markus
AU - Kubsch, Bastian
AU - Hatahet, Lina
AU - Alkoby, Dudu
AU - Rimon, Abraham
AU - Padan, Etana
AU - Fendler, Klaus
PY - 2013/8/23
Y1 - 2013/8/23
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84883171052&partnerID=8YFLogxK
U2 - 10.1074/jbc.M113.484071
DO - 10.1074/jbc.M113.484071
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C2 - 23836890
AN - SCOPUS:84883171052
SN - 0021-9258
VL - 288
SP - 24666
EP - 24675
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 34
ER -