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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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A, fit of the sodium jump-induced peak currents of V254C NhaA to the asymmetric kinetic model. Simultaneous fit of the pH dependences was at indicated sodium concentrations, and the sodium dependences was at indicated pH values of V254C NhaA in both transport directions. The solid line is a fit to the asymmetric kinetic model described in the text. The kinetic parameters obtained by the fit are as follows: KDNa = 2.7 ± 0.6 mm, pKa = 8.9 ± 0.1 mm, k2−/k1+ = 7.0, and k1+/k1− = 3.4. Data and conditions as in Fig. 2 and Table 1. B, energy diagram of WT NhaA and V254C NhaA in a simplified representation. CS is the transporter in the substrate-bound form, S = Na+ (red) or S = H+ (blue). The subscript i (inside) marks the state of the transporter opened to the cytoplasm, and the subscript o (outside) marks the state of the transporter opened to the periplasm. The half-cycles are graphical representations of the energy barriers between the inside opened and the outside opened conformation of the transporter in the sodium-bound form (red) or in the proton-bound form (blue). ΔΔG* is the difference of the activation enthalpies of the transitions states. ΔG is the free enthalpy difference of the inside opened and the outside opened conformation.
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Figure 5: A, fit of the sodium jump-induced peak currents of V254C NhaA to the asymmetric kinetic model. Simultaneous fit of the pH dependences was at indicated sodium concentrations, and the sodium dependences was at indicated pH values of V254C NhaA in both transport directions. The solid line is a fit to the asymmetric kinetic model described in the text. The kinetic parameters obtained by the fit are as follows: KDNa = 2.7 ± 0.6 mm, pKa = 8.9 ± 0.1 mm, k2−/k1+ = 7.0, and k1+/k1− = 3.4. Data and conditions as in Fig. 2 and Table 1. B, energy diagram of WT NhaA and V254C NhaA in a simplified representation. CS is the transporter in the substrate-bound form, S = Na+ (red) or S = H+ (blue). The subscript i (inside) marks the state of the transporter opened to the cytoplasm, and the subscript o (outside) marks the state of the transporter opened to the periplasm. The half-cycles are graphical representations of the energy barriers between the inside opened and the outside opened conformation of the transporter in the sodium-bound form (red) or in the proton-bound form (blue). ΔΔG* is the difference of the activation enthalpies of the transitions states. ΔG is the free enthalpy difference of the inside opened and the outside opened conformation.

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)

A, fit of the sodium jump-induced peak currents of V254C NhaA to the asymmetric kinetic model. Simultaneous fit of the pH dependences was at indicated sodium concentrations, and the sodium dependences was at indicated pH values of V254C NhaA in both transport directions. The solid line is a fit to the asymmetric kinetic model described in the text. The kinetic parameters obtained by the fit are as follows: KDNa = 2.7 ± 0.6 mm, pKa = 8.9 ± 0.1 mm, k2−/k1+ = 7.0, and k1+/k1− = 3.4. Data and conditions as in Fig. 2 and Table 1. B, energy diagram of WT NhaA and V254C NhaA in a simplified representation. CS is the transporter in the substrate-bound form, S = Na+ (red) or S = H+ (blue). The subscript i (inside) marks the state of the transporter opened to the cytoplasm, and the subscript o (outside) marks the state of the transporter opened to the periplasm. The half-cycles are graphical representations of the energy barriers between the inside opened and the outside opened conformation of the transporter in the sodium-bound form (red) or in the proton-bound form (blue). ΔΔG* is the difference of the activation enthalpies of the transitions states. ΔG is the free enthalpy difference of the inside opened and the outside opened conformation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: A, fit of the sodium jump-induced peak currents of V254C NhaA to the asymmetric kinetic model. Simultaneous fit of the pH dependences was at indicated sodium concentrations, and the sodium dependences was at indicated pH values of V254C NhaA in both transport directions. The solid line is a fit to the asymmetric kinetic model described in the text. The kinetic parameters obtained by the fit are as follows: KDNa = 2.7 ± 0.6 mm, pKa = 8.9 ± 0.1 mm, k2−/k1+ = 7.0, and k1+/k1− = 3.4. Data and conditions as in Fig. 2 and Table 1. B, energy diagram of WT NhaA and V254C NhaA in a simplified representation. CS is the transporter in the substrate-bound form, S = Na+ (red) or S = H+ (blue). The subscript i (inside) marks the state of the transporter opened to the cytoplasm, and the subscript o (outside) marks the state of the transporter opened to the periplasm. The half-cycles are graphical representations of the energy barriers between the inside opened and the outside opened conformation of the transporter in the sodium-bound form (red) or in the proton-bound form (blue). ΔΔG* is the difference of the activation enthalpies of the transitions states. ΔG is the free enthalpy difference of the inside opened and the outside opened conformation.
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