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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,22Na uptake of A167P NhaA reconstituted in liposomes in comparison with WT NhaA with and without (+valinomycin), a membrane potential. NhaA proteoliposomes were reconstituted, and ΔpH-driven 22Na uptake was determined as described previously (4). All experiments were repeated at least twice with practically identical results. B and C, pH dependences of transient currents obtained with A167P proteoliposomes after a Na+ concentration jump. B, transient currents after 100 mm Na+ concentration jump at indicated pH values. The transient currents were artifact corrected as described under “Experimental Procedures.” C, normalized peak currents obtained with A167P proteoliposomes (in red) at indicated pH values after a 10 mm or 100 mm Na+ concentration jump. For comparison, the normalized peak currents obtained with WT NhaA proteoliposomes are shown in black. The graph shows average values from recordings using three individual sensors and the corresponding S.D. Currents were normalized as described under “Experimental Procedures.” For the Na+ concentration jumps, activating solutions containing 10 mm NaCl and 290 mm KCl or 100 mm NaCl and 200 mm KCl titrated to the indicated pH values when HCl or Tris were used. The nonactivating solutions contained 300 mm KCl instead. In addition, all buffers contained 5 mm MgCl2, 25 mm Tris, 25 mm MOPS, 25 mm MES, and 1 mm DTT.
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Figure 3: A,22Na uptake of A167P NhaA reconstituted in liposomes in comparison with WT NhaA with and without (+valinomycin), a membrane potential. NhaA proteoliposomes were reconstituted, and ΔpH-driven 22Na uptake was determined as described previously (4). All experiments were repeated at least twice with practically identical results. B and C, pH dependences of transient currents obtained with A167P proteoliposomes after a Na+ concentration jump. B, transient currents after 100 mm Na+ concentration jump at indicated pH values. The transient currents were artifact corrected as described under “Experimental Procedures.” C, normalized peak currents obtained with A167P proteoliposomes (in red) at indicated pH values after a 10 mm or 100 mm Na+ concentration jump. For comparison, the normalized peak currents obtained with WT NhaA proteoliposomes are shown in black. The graph shows average values from recordings using three individual sensors and the corresponding S.D. Currents were normalized as described under “Experimental Procedures.” For the Na+ concentration jumps, activating solutions containing 10 mm NaCl and 290 mm KCl or 100 mm NaCl and 200 mm KCl titrated to the indicated pH values when HCl or Tris were used. The nonactivating solutions contained 300 mm KCl instead. In addition, all buffers contained 5 mm MgCl2, 25 mm Tris, 25 mm MOPS, 25 mm MES, and 1 mm DTT.

Mentions: Table lists the kinetic parameters of V254C NhaA, A167P NhaA, H225R NhaA, and ΔP45-N58 NhaA. The KmNa values were determined at indicated pH values by the hyperbolic fit to the Na+ dependencies. For the Na+ concentration, jumps activating solutions containing x mm NaCl and (300 − x) mm KCl titrated to the indicated pH values with Tris were used. The nonactivating solutions contained 300 mm KCl instead. In addition, all buffers contained 5 mm MgCl2, 25 mm Tris, 25 mm MOPS, 25 mm MES, and 1 mm DTT. For comparison, Vmax, the average of the saturation values of the hyperbolic fit is shown. The pH of maximal peak current pHopt was taken from the Voigt fit to the pH dependencies after a 100 mm Na+ concentration jump (Figs. 2 and 3C).


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,22Na uptake of A167P NhaA reconstituted in liposomes in comparison with WT NhaA with and without (+valinomycin), a membrane potential. NhaA proteoliposomes were reconstituted, and ΔpH-driven 22Na uptake was determined as described previously (4). All experiments were repeated at least twice with practically identical results. B and C, pH dependences of transient currents obtained with A167P proteoliposomes after a Na+ concentration jump. B, transient currents after 100 mm Na+ concentration jump at indicated pH values. The transient currents were artifact corrected as described under “Experimental Procedures.” C, normalized peak currents obtained with A167P proteoliposomes (in red) at indicated pH values after a 10 mm or 100 mm Na+ concentration jump. For comparison, the normalized peak currents obtained with WT NhaA proteoliposomes are shown in black. The graph shows average values from recordings using three individual sensors and the corresponding S.D. Currents were normalized as described under “Experimental Procedures.” For the Na+ concentration jumps, activating solutions containing 10 mm NaCl and 290 mm KCl or 100 mm NaCl and 200 mm KCl titrated to the indicated pH values when HCl or Tris were used. The nonactivating solutions contained 300 mm KCl instead. In addition, all buffers contained 5 mm MgCl2, 25 mm Tris, 25 mm MOPS, 25 mm MES, and 1 mm DTT.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: A,22Na uptake of A167P NhaA reconstituted in liposomes in comparison with WT NhaA with and without (+valinomycin), a membrane potential. NhaA proteoliposomes were reconstituted, and ΔpH-driven 22Na uptake was determined as described previously (4). All experiments were repeated at least twice with practically identical results. B and C, pH dependences of transient currents obtained with A167P proteoliposomes after a Na+ concentration jump. B, transient currents after 100 mm Na+ concentration jump at indicated pH values. The transient currents were artifact corrected as described under “Experimental Procedures.” C, normalized peak currents obtained with A167P proteoliposomes (in red) at indicated pH values after a 10 mm or 100 mm Na+ concentration jump. For comparison, the normalized peak currents obtained with WT NhaA proteoliposomes are shown in black. The graph shows average values from recordings using three individual sensors and the corresponding S.D. Currents were normalized as described under “Experimental Procedures.” For the Na+ concentration jumps, activating solutions containing 10 mm NaCl and 290 mm KCl or 100 mm NaCl and 200 mm KCl titrated to the indicated pH values when HCl or Tris were used. The nonactivating solutions contained 300 mm KCl instead. In addition, all buffers contained 5 mm MgCl2, 25 mm Tris, 25 mm MOPS, 25 mm MES, and 1 mm DTT.
Mentions: Table lists the kinetic parameters of V254C NhaA, A167P NhaA, H225R NhaA, and ΔP45-N58 NhaA. The KmNa values were determined at indicated pH values by the hyperbolic fit to the Na+ dependencies. For the Na+ concentration, jumps activating solutions containing x mm NaCl and (300 − x) mm KCl titrated to the indicated pH values with Tris were used. The nonactivating solutions contained 300 mm KCl instead. In addition, all buffers contained 5 mm MgCl2, 25 mm Tris, 25 mm MOPS, 25 mm MES, and 1 mm DTT. For comparison, Vmax, the average of the saturation values of the hyperbolic fit is shown. The pH of maximal peak current pHopt was taken from the Voigt fit to the pH dependencies after a 100 mm Na+ concentration jump (Figs. 2 and 3C).

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