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

Mager T, Braner M, Kubsch B, Hatahet L, Alkoby D, Rimon A, Padan E, Fendler K - J. Biol. Chem. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Biophysik, 60438 Frankfurt/Main, Germany.

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.

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Simultaneous fit of the pH dependences at indicated sodium concentrations and the sodium dependences at indicated pH values to the kinetic model. The graphs show sodium jump-induced peak currents using ΔP45-N58 NhaA RSO proteoliposomes (A and B), ΔP45-N58 NhaA ISO membrane vesicles (C and D), H225R NhaA RSO proteoliposomes (E and F), and A167P RSO proteoliposomes (G and H). Data and conditions are as in Fig. 2 (pH dependences) and Table 1 (Na+ dependences). The solid line is a fit to the kinetic model described in the text. The kinetic parameters determined by the fit are shown in the figure.
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Figure 4: Simultaneous fit of the pH dependences at indicated sodium concentrations and the sodium dependences at indicated pH values to the kinetic model. The graphs show sodium jump-induced peak currents using ΔP45-N58 NhaA RSO proteoliposomes (A and B), ΔP45-N58 NhaA ISO membrane vesicles (C and D), H225R NhaA RSO proteoliposomes (E and F), and A167P RSO proteoliposomes (G and H). Data and conditions are as in Fig. 2 (pH dependences) and Table 1 (Na+ dependences). The solid line is a fit to the kinetic model described in the text. The kinetic parameters determined by the fit are shown in the figure.

Mentions: The fitted parameters determined for the different mutants and the WT are given in Figs. 4 and 5 and are summarized for comparison in Table 2. We observe three different phenotypes as follows: pK-shifted variants G338S and H225R, low Na+ affinity variants H225R and A167P, but also variants with modified conformation dynamics A167P and V254C. The latter is important because it demonstrates that a simple kinetic characterization based on KmNa and Vmax values can be misleading.


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

Mager T, Braner M, Kubsch B, Hatahet L, Alkoby D, Rimon A, Padan E, Fendler K - J. Biol. Chem. (2013)

Simultaneous fit of the pH dependences at indicated sodium concentrations and the sodium dependences at indicated pH values to the kinetic model. The graphs show sodium jump-induced peak currents using ΔP45-N58 NhaA RSO proteoliposomes (A and B), ΔP45-N58 NhaA ISO membrane vesicles (C and D), H225R NhaA RSO proteoliposomes (E and F), and A167P RSO proteoliposomes (G and H). Data and conditions are as in Fig. 2 (pH dependences) and Table 1 (Na+ dependences). The solid line is a fit to the kinetic model described in the text. The kinetic parameters determined by the fit are shown in the figure.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750164&req=5

Figure 4: Simultaneous fit of the pH dependences at indicated sodium concentrations and the sodium dependences at indicated pH values to the kinetic model. The graphs show sodium jump-induced peak currents using ΔP45-N58 NhaA RSO proteoliposomes (A and B), ΔP45-N58 NhaA ISO membrane vesicles (C and D), H225R NhaA RSO proteoliposomes (E and F), and A167P RSO proteoliposomes (G and H). Data and conditions are as in Fig. 2 (pH dependences) and Table 1 (Na+ dependences). The solid line is a fit to the kinetic model described in the text. The kinetic parameters determined by the fit are shown in the figure.
Mentions: The fitted parameters determined for the different mutants and the WT are given in Figs. 4 and 5 and are summarized for comparison in Table 2. We observe three different phenotypes as follows: pK-shifted variants G338S and H225R, low Na+ affinity variants H225R and A167P, but also variants with modified conformation dynamics A167P and V254C. The latter is important because it demonstrates that a simple kinetic characterization based on KmNa and Vmax values can be misleading.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Biophysik, 60438 Frankfurt/Main, Germany.

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.

Show MeSH
Related in: MedlinePlus