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NhaA Na+/H+ antiporter mutants that hardly react to the membrane potential.

Alkoby D, Rimon A, Budak M, Burdak M, Patino-Ruiz M, Călinescu O, Fendler K, Padan E - PLoS ONE (2014)

Bottom Line: The selected mutants, A167P and F267C are described in detail.Surprising for an electrogenic secondary transporter, and opposed to wild type NhaA, the rates of A167P and F267C are almost indifferent to membrane potential.Detailed kinetic analysis reveals that in both mutants the rate limiting step of the cation exchange cycle is changed from an electrogenic to an electroneutral reaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University, Jerusalem, Israel.

ABSTRACT
pH and Na+ homeostasis in all cells requires Na+/H+ antiporters. The crystal structure, obtained at pH 4, of NhaA, the main antiporter of Escherichia coli, has provided general insights into an antiporter mechanism and its unique pH regulation. Here, we describe a general method to select various NhaA mutants from a library of randomly mutagenized NhaA. The selected mutants, A167P and F267C are described in detail. Both mutants are expressed in Escherichia coli EP432 cells at 70-95% of the wild type but grow on selective medium only at neutral pH, A167P on Li+ (0.1 M) and F267C on Na+ (0.6 M). Surprising for an electrogenic secondary transporter, and opposed to wild type NhaA, the rates of A167P and F267C are almost indifferent to membrane potential. Detailed kinetic analysis reveals that in both mutants the rate limiting step of the cation exchange cycle is changed from an electrogenic to an electroneutral reaction.

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pH dependence of the Na+, Li+/H+ antiporter activity in everted membrane vesicles of variant A167P.Everted membrane vesicles were prepared from EP432 cells grown in LBK (pH 7) and expressing WT (□) or A167P (Δ). The Na+/H+ and Li+/H+ antiporter activity was determined in the presence of 10 mM NaCl (filled symbols) or 10 mM LiCl (open symbols) at the indicated pH values, using acridine orange fluorescence to monitor ΔpH. Results are expressed in % of maximal dequenching of the fluorescence due to cation addition. All experiments were repeated at least three times with nearly identical results.
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pone-0093200-g003: pH dependence of the Na+, Li+/H+ antiporter activity in everted membrane vesicles of variant A167P.Everted membrane vesicles were prepared from EP432 cells grown in LBK (pH 7) and expressing WT (□) or A167P (Δ). The Na+/H+ and Li+/H+ antiporter activity was determined in the presence of 10 mM NaCl (filled symbols) or 10 mM LiCl (open symbols) at the indicated pH values, using acridine orange fluorescence to monitor ΔpH. Results are expressed in % of maximal dequenching of the fluorescence due to cation addition. All experiments were repeated at least three times with nearly identical results.

Mentions: We have previously shown [29], [30] that certain mutations in NhaA only affect the apparent Km, and not the pH dependence of the exchanger. These mutants show an altered pH dependence at non-saturating Na+ concentrations but a pH dependence similar to the wild type at saturating Na+. By contrast, other mutations retain an altered pH dependence at both saturating and non-saturating Na+ concentrations, irrespective of whether the Na+ affinity is changed or not from the WT. Therefore, the pH profile of the antiporter activity in membrane vesicles isolated from E. coli EP432/A167P was measured at saturating- concentrations of the cations (Fig. 3). The pH dependence of both Na+/H+ and Li+/H+ antiport activity of the variants was shifted by about 1 pH unit to the alkaline side as compared to the WT.


NhaA Na+/H+ antiporter mutants that hardly react to the membrane potential.

Alkoby D, Rimon A, Budak M, Burdak M, Patino-Ruiz M, Călinescu O, Fendler K, Padan E - PLoS ONE (2014)

pH dependence of the Na+, Li+/H+ antiporter activity in everted membrane vesicles of variant A167P.Everted membrane vesicles were prepared from EP432 cells grown in LBK (pH 7) and expressing WT (□) or A167P (Δ). The Na+/H+ and Li+/H+ antiporter activity was determined in the presence of 10 mM NaCl (filled symbols) or 10 mM LiCl (open symbols) at the indicated pH values, using acridine orange fluorescence to monitor ΔpH. Results are expressed in % of maximal dequenching of the fluorescence due to cation addition. All experiments were repeated at least three times with nearly identical results.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3974702&req=5

pone-0093200-g003: pH dependence of the Na+, Li+/H+ antiporter activity in everted membrane vesicles of variant A167P.Everted membrane vesicles were prepared from EP432 cells grown in LBK (pH 7) and expressing WT (□) or A167P (Δ). The Na+/H+ and Li+/H+ antiporter activity was determined in the presence of 10 mM NaCl (filled symbols) or 10 mM LiCl (open symbols) at the indicated pH values, using acridine orange fluorescence to monitor ΔpH. Results are expressed in % of maximal dequenching of the fluorescence due to cation addition. All experiments were repeated at least three times with nearly identical results.
Mentions: We have previously shown [29], [30] that certain mutations in NhaA only affect the apparent Km, and not the pH dependence of the exchanger. These mutants show an altered pH dependence at non-saturating Na+ concentrations but a pH dependence similar to the wild type at saturating Na+. By contrast, other mutations retain an altered pH dependence at both saturating and non-saturating Na+ concentrations, irrespective of whether the Na+ affinity is changed or not from the WT. Therefore, the pH profile of the antiporter activity in membrane vesicles isolated from E. coli EP432/A167P was measured at saturating- concentrations of the cations (Fig. 3). The pH dependence of both Na+/H+ and Li+/H+ antiport activity of the variants was shifted by about 1 pH unit to the alkaline side as compared to the WT.

Bottom Line: The selected mutants, A167P and F267C are described in detail.Surprising for an electrogenic secondary transporter, and opposed to wild type NhaA, the rates of A167P and F267C are almost indifferent to membrane potential.Detailed kinetic analysis reveals that in both mutants the rate limiting step of the cation exchange cycle is changed from an electrogenic to an electroneutral reaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University, Jerusalem, Israel.

ABSTRACT
pH and Na+ homeostasis in all cells requires Na+/H+ antiporters. The crystal structure, obtained at pH 4, of NhaA, the main antiporter of Escherichia coli, has provided general insights into an antiporter mechanism and its unique pH regulation. Here, we describe a general method to select various NhaA mutants from a library of randomly mutagenized NhaA. The selected mutants, A167P and F267C are described in detail. Both mutants are expressed in Escherichia coli EP432 cells at 70-95% of the wild type but grow on selective medium only at neutral pH, A167P on Li+ (0.1 M) and F267C on Na+ (0.6 M). Surprising for an electrogenic secondary transporter, and opposed to wild type NhaA, the rates of A167P and F267C are almost indifferent to membrane potential. Detailed kinetic analysis reveals that in both mutants the rate limiting step of the cation exchange cycle is changed from an electrogenic to an electroneutral reaction.

Show MeSH
Related in: MedlinePlus