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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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Related in: MedlinePlus

A, helices V, VIII, IX. and XI in the structure of NhaA with the modified amino acid residues indicated in yellow. The two aspartates of the putative cation-binding site (Asp-163 and Asp-164) are shown in blue and red. The β-sheet P45-N58 is drawn in yellow. The red dotted lines indicate the trajectories from the mutated residues to the cation-binding site. B, kinetic model for Na+/H+ antiport. In the “forward mode,” the outside directed transporter Co binds H+ (this could be one or two H+ ions, and for simplicity only one is considered in the model) from the periplasm (Ho), performs a conformational transition to the inward directed form Ci, and releases H+ to the cytoplasm. Subsequently, Na+ is bound from the cytoplasm (Nai) and released to the periplasm.
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Figure 1: A, helices V, VIII, IX. and XI in the structure of NhaA with the modified amino acid residues indicated in yellow. The two aspartates of the putative cation-binding site (Asp-163 and Asp-164) are shown in blue and red. The β-sheet P45-N58 is drawn in yellow. The red dotted lines indicate the trajectories from the mutated residues to the cation-binding site. B, kinetic model for Na+/H+ antiport. In the “forward mode,” the outside directed transporter Co binds H+ (this could be one or two H+ ions, and for simplicity only one is considered in the model) from the periplasm (Ho), performs a conformational transition to the inward directed form Ci, and releases H+ to the cytoplasm. Subsequently, Na+ is bound from the cytoplasm (Nai) and released to the periplasm.

Mentions: To investigate the effect of mutations remote from the active site on the pH profile, we functionally characterized the mutants V254C NhaA and H225R NhaA (Fig. 1A). Val-254 is located at the cytoplasmic end of helix IX, which is part of the postulated pH sensor region. His-225 is in helix VIII, which is flanking the periplasmic funnel. These mutants showed an altered pH dependence at preceding biochemical investigations (7, 12–15).


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, helices V, VIII, IX. and XI in the structure of NhaA with the modified amino acid residues indicated in yellow. The two aspartates of the putative cation-binding site (Asp-163 and Asp-164) are shown in blue and red. The β-sheet P45-N58 is drawn in yellow. The red dotted lines indicate the trajectories from the mutated residues to the cation-binding site. B, kinetic model for Na+/H+ antiport. In the “forward mode,” the outside directed transporter Co binds H+ (this could be one or two H+ ions, and for simplicity only one is considered in the model) from the periplasm (Ho), performs a conformational transition to the inward directed form Ci, and releases H+ to the cytoplasm. Subsequently, Na+ is bound from the cytoplasm (Nai) and released to the periplasm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: A, helices V, VIII, IX. and XI in the structure of NhaA with the modified amino acid residues indicated in yellow. The two aspartates of the putative cation-binding site (Asp-163 and Asp-164) are shown in blue and red. The β-sheet P45-N58 is drawn in yellow. The red dotted lines indicate the trajectories from the mutated residues to the cation-binding site. B, kinetic model for Na+/H+ antiport. In the “forward mode,” the outside directed transporter Co binds H+ (this could be one or two H+ ions, and for simplicity only one is considered in the model) from the periplasm (Ho), performs a conformational transition to the inward directed form Ci, and releases H+ to the cytoplasm. Subsequently, Na+ is bound from the cytoplasm (Nai) and released to the periplasm.
Mentions: To investigate the effect of mutations remote from the active site on the pH profile, we functionally characterized the mutants V254C NhaA and H225R NhaA (Fig. 1A). Val-254 is located at the cytoplasmic end of helix IX, which is part of the postulated pH sensor region. His-225 is in helix VIII, which is flanking the periplasmic funnel. These mutants showed an altered pH dependence at preceding biochemical investigations (7, 12–15).

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