Eukaryotic Na؉ /H ؉ exchangers are transmembrane proteins that are vital for cellular homeostasis and play key roles in pathological conditions such as cancer and heart diseases. Using the crystal structure of the Na ؉ /H ؉ antiporter from Escherichia coli (EcNhaA) as a template, we predicted the three-dimensional structure of human Na ؉ /H ؉ exchanger 1 (NHE1). Modeling was particularly challenging because of the extremely low sequence identity between these proteins, but the model structure is supported by evolutionary conservation analysis and empirical data. It also revealed the location of the binding site of NHE inhibitors; which we validated by conducting mutagenesis studies with EcNhaA and its specific inhibitor 2-aminoperimidine. The model structure features a cluster of titratable residues that are evolutionarily conserved and are located in a conserved region in the center of the membrane; we suggest that they are involved in the cation binding and translocation. We also suggest a hypothetical alternating-access mechanism that involves conformational changes.

Na(+)/H(+) antiporters are ubiquitous membrane proteins that are involved in homeostasis of H(+) and Na(+) throughout the biological kingdom. Corroborating their role in pH homeostasis, many of the Na(+)/H(+) antiporter proteins are regulated directly by pH. The pH regulation of NhaA, the Escherichia coli Na(+)/H(+) antiporter (EcNhaA), as of other, both eukaryotic and prokaryotic Na(+)/H(+) antiporters, involves a pH sensor and conformational changes in different parts of the protein that transduce the pH signal into a change in activity. Thus, residues that affect the pH response, the translocation or both activities cluster in separate domains along the antiporter molecules. Importantly, in the NhaA family, these domains are conserved. Helix-packing model of EcNhaA based on cross-linking data suggests, that in the three dimensional structure of NhaA, residues that affect the pH response may be in close proximity, forming a single pH sensitive domain. Therefore, it is suggested that, despite considerable differences in the primary structure of the antiporters from the bacterial NhaA to the mammalian NHEs, their three-dimensional architectures are conserved. Test of this possibility awaits the atomic resolution of the 3D structure of the antiporters.

). The NhaA crystal structure has provided insights into the pH-regulated mechanism of antiporter action and opened up new in silico and in situ avenues of research. The monomer is the functional unit of NhaA yet the dimer is essential for the stability of the antiporter under extreme stress conditions. Ionizable residues of NhaA that strongly interact electrostatically are organized in a transmembrane fashion in accordance with the functional organization of the cation-binding site, 'pH sensor', the pH transduction pathway and the pH-induced conformational changes. Remarkably, NhaA contains an inverted topology motive of transmembrane segments, which are interrupted by extended mid-membrane chains that have since been found to vary in other ion-transport proteins. This novel structural fold creates a delicately balanced electrostatic environment in the middle of the membrane, which might be essential for ion binding and translocation. Based on the crystal structure of NhaA, a model structure of the human Na + /H + exchanger (NHE1) was constructed, paving the way to a rational drug design.

Vibrio cholerae, the causative agent of cholera, is a normal inhabitant of aquatic environments, where it survives in a wide range of conditions of pH and salinity. In this work, we investigated the role of three Na ؉ /H ؉ antiporters on the survival of V. cholerae in a saline environment. We have previously cloned the Vc-nhaA gene encoding the V. cholerae homolog of Escherichia coli. Here we identified two additional antiporter genes, designated Vc-nhaB and Vc-nhaD, encoding two putative proteins of 530 and 477 residues, respectively, highly homologous to the respective antiporters of Vibrio species and E. coli. We showed that both Vc-NhaA and Vc-NhaB confer Na ؉ resistance and that Vc-NhaA displays an antiport activity in E. coli, which is similar in magnitude, kinetic parameters, and pH regulation to that of E. coli NhaA. To determine the roles of the Na ؉ /H ؉ antiporters in V. cholerae, we constructed nhaA, nhaB, and nhaD mutants (single, double, and triple mutants). In contrast to E. coli, the inactivation of the three putative antiporter genes (Vc-nhaABD) in V. cholerae did not alter the bacterial exponential growth in the presence of high Na ؉ concentrations and had only a slight effect in the stationary phase. In contrast, a pronounced and similar Li ؉ -sensitive phenotype was found with all mutants lacking Vc-nhaA during the exponential phase of growth and also with the triple mutant in the stationary phase of growth. By using 2-n-nonyl-4-hydroxyquinoline N-oxide, a specific inhibitor of the electron-transport-linked Na ؉ pump NADH-quinone oxidoreductase (NQR), we determined that in the absence of NQR activity, the Vc-NhaA Na ؉ /H ؉ antiporter activity becomes essential for the resistance of V. cholerae to Na ؉ at alkaline pH. Since the ion pump NQR is Na ؉ specific, we suggest that its activity masks the Na ؉ /H ؉ but not the Li ؉ /H ؉ antiporter activities. Our results indicate that the Na ؉ resistance of the human pathogen V. cholerae requires a complex molecular system involving multiple antiporters and the NQR pump.

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