Abstract
Na+/H+ antiporters are located in the cytoplasmic and intracellular membranes and play crucial roles in regulating intracellular pH, Na+, and volume. The NhaA antiporter of Escherichia coli is the best studied member of the Na+/H+ exchanger family and a model system for all related Na+/H+ exchangers, including eukaryotic representatives. Several amino acid residues are important for the transport activity of NhaA, including Lys-300, a residue that has recently been proposed to carry one of the two H+ ions that NhaA exchanges for one Na+ ion during one transport cycle. Here, we sought to characterize the effects of mutating Lys-300 of NhaA to amino acid residues containing side chains of different polarity and length (i.e. Ala, Arg, Cys, His, Glu, and Leu) on transporter stability and function. Salt resistance assays, acridine-orange fluorescence dequenching, solid supported membrane-based electrophysiology, and differential scanning fluorometry were used to characterize Na+ and H+ transport, charge translocation, and thermal stability of the different variants. These studies revealed that NhaA could still perform electrogenic Na+/H+ exchange even in the absence of a protonatable residue at the Lys-300 position. However, all mutants displayed lower thermal stability and reduced ion transport activity compared with the wild-type enzyme, indicating the critical importance of Lys-300 for optimal NhaA structural stability and function. On the basis of these experimental data, we propose a tentative mechanism integrating the functional and structural role of Lys-300.
Highlights
Na؉/H؉ antiporters are located in the cytoplasmic and intracellular membranes and play crucial roles in regulating intracellular pH, Na؉, and volume
To characterize the mutants with respect to expression, growth, and antiporter activity in everted isolated membrane vesicles, the mutant plasmids were transformed into EP432, an E. coli strain that lacks the two specific Naϩ/Hϩ antiporters NhaA and NhaB [35]
We found a reduced thermal stability of the variants that depended on the character of the amino acid residue in position
Summary
Na؉/H؉ antiporters are located in the cytoplasmic and intracellular membranes and play crucial roles in regulating intracellular pH, Na؉, and volume. We sought to characterize the effects of mutating Lys-300 of NhaA to amino acid residues containing side chains of different polarity and length (i.e. Ala, Arg, Cys, His, Glu, and Leu) on transporter stability and function. Acridine-orange fluorescence dequenching, solid supported membrane-based electrophysiology, and differential scanning fluorometry were used to characterize Na؉ and H؉ transport, charge translocation, and thermal stability of the different variants. These studies revealed that NhaA could still perform electrogenic Na؉/H؉ exchange even in the absence of a protonatable residue at the Lys-300 position. Naϩ/Hϩ antiporters, playing a primary role in these homeostatic processes, are located in the cytoplasmic and intracellular membranes of cells. Homologues of EcNhaA4 (NhaA), the main Escherichia coli Naϩ, Liϩ/Hϩ antiporter, have recently been implicated in the virulence of pathogenic bacteria [5, 6] and in human essential hypertension [7] as well as diabetes [8]
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