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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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pH dependences of transient currents obtained with H225R NhaA, V254C NhaA, and ΔP45-N58 NhaA after a Na+ concentration jump. Shown are the normalized peak currents at indicated pH values after a 10 or 100 mm Na+ concentration jump. As a guide to the eye, the pH dependences were fitted with a Voigt function. Data obtained with RSO proteoliposomes of H225R NhaA (A), V254C NhaA (B), and ΔP45-N58 NhaA (D) as well as data obtained with ISO membrane vesicles of V254C NhaA (C) and ΔP45-N58 NhaA (E) are shown in red. For comparison, the WT NhaA pH dependences are included in black (taken from Ref. 9). In the upper right panel, a graphic representation of the transport modes of NhaA in the bacterial cell and under experimental conditions is shown. The graphs show 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 with 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 2: pH dependences of transient currents obtained with H225R NhaA, V254C NhaA, and ΔP45-N58 NhaA after a Na+ concentration jump. Shown are the normalized peak currents at indicated pH values after a 10 or 100 mm Na+ concentration jump. As a guide to the eye, the pH dependences were fitted with a Voigt function. Data obtained with RSO proteoliposomes of H225R NhaA (A), V254C NhaA (B), and ΔP45-N58 NhaA (D) as well as data obtained with ISO membrane vesicles of V254C NhaA (C) and ΔP45-N58 NhaA (E) are shown in red. For comparison, the WT NhaA pH dependences are included in black (taken from Ref. 9). In the upper right panel, a graphic representation of the transport modes of NhaA in the bacterial cell and under experimental conditions is shown. The graphs show 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 with 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: To perform a kinetic analysis of the transporter activity of ΔP45-N58, V254C, H225R, and A167P NhaA, right-side out (RSO) proteoliposomes, exposing the periplasmic side of the transporter to the outside, were prepared and investigated using SSM-based electrophysiology. Note that NhaA spontaneously adopts an RSO orientation in proteoliposomes (23). V254C NhaA and ΔP45-N58 NhaA were also studied in the opposite transport direction using ISO membrane vesicles derived from bacteria overexpressing the corresponding mutant proteins and exposing the cytoplasmic side to the outside (Fig. 2A, right panel) (9). Membrane vesicles prepared by French press cell disruption have a preferential ISO orientation (24). A complete ISO orientation of the vesicles was further ensured using an extraction procedure with nickel-nitrilotriacetic acid-tagged beads (21). Transport was initiated by a rapid Na+ concentration jump. The ensuing transporter activity generates a transient current. Its peak value is a reliable estimate of the steady-state transport activity of NhaA (23). By applying an inwardly directed Na+ gradient to the RSO proteoliposomes, transport against physiological transport direction (reverse mode) was investigated (Fig. 2, A, B, and D). Transport in physiological transport direction (forward mode) was investigated by applying an inwardly directed Na+ gradient (Na+ inside = 0) to ISO membrane vesicles (Fig. 2, C and E).


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)

pH dependences of transient currents obtained with H225R NhaA, V254C NhaA, and ΔP45-N58 NhaA after a Na+ concentration jump. Shown are the normalized peak currents at indicated pH values after a 10 or 100 mm Na+ concentration jump. As a guide to the eye, the pH dependences were fitted with a Voigt function. Data obtained with RSO proteoliposomes of H225R NhaA (A), V254C NhaA (B), and ΔP45-N58 NhaA (D) as well as data obtained with ISO membrane vesicles of V254C NhaA (C) and ΔP45-N58 NhaA (E) are shown in red. For comparison, the WT NhaA pH dependences are included in black (taken from Ref. 9). In the upper right panel, a graphic representation of the transport modes of NhaA in the bacterial cell and under experimental conditions is shown. The graphs show 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 with 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

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

Figure 2: pH dependences of transient currents obtained with H225R NhaA, V254C NhaA, and ΔP45-N58 NhaA after a Na+ concentration jump. Shown are the normalized peak currents at indicated pH values after a 10 or 100 mm Na+ concentration jump. As a guide to the eye, the pH dependences were fitted with a Voigt function. Data obtained with RSO proteoliposomes of H225R NhaA (A), V254C NhaA (B), and ΔP45-N58 NhaA (D) as well as data obtained with ISO membrane vesicles of V254C NhaA (C) and ΔP45-N58 NhaA (E) are shown in red. For comparison, the WT NhaA pH dependences are included in black (taken from Ref. 9). In the upper right panel, a graphic representation of the transport modes of NhaA in the bacterial cell and under experimental conditions is shown. The graphs show 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 with 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: To perform a kinetic analysis of the transporter activity of ΔP45-N58, V254C, H225R, and A167P NhaA, right-side out (RSO) proteoliposomes, exposing the periplasmic side of the transporter to the outside, were prepared and investigated using SSM-based electrophysiology. Note that NhaA spontaneously adopts an RSO orientation in proteoliposomes (23). V254C NhaA and ΔP45-N58 NhaA were also studied in the opposite transport direction using ISO membrane vesicles derived from bacteria overexpressing the corresponding mutant proteins and exposing the cytoplasmic side to the outside (Fig. 2A, right panel) (9). Membrane vesicles prepared by French press cell disruption have a preferential ISO orientation (24). A complete ISO orientation of the vesicles was further ensured using an extraction procedure with nickel-nitrilotriacetic acid-tagged beads (21). Transport was initiated by a rapid Na+ concentration jump. The ensuing transporter activity generates a transient current. Its peak value is a reliable estimate of the steady-state transport activity of NhaA (23). By applying an inwardly directed Na+ gradient to the RSO proteoliposomes, transport against physiological transport direction (reverse mode) was investigated (Fig. 2, A, B, and D). Transport in physiological transport direction (forward mode) was investigated by applying an inwardly directed Na+ gradient (Na+ inside = 0) to ISO membrane vesicles (Fig. 2, C and E).

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