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.

We have previously shown that the activity of NhaA is regulated by pH and found mutations that affect dramatically the pH dependence of the rate but not the K(m) (for Na(+) and Li(+)) of NhaA. In the present work, we found that helix IV is involved both in ion translocation as well as in pH regulation of NhaA. Two novel types of NhaA mutants were found clustered in trans membrane segment (TMS) IV: One type (D133C, T132C, and P129L) affects the apparent K(m) of NhaA to the cations with no significant effect on the pH profile of the antiporter; no shift of the pH profile was found when the activity of these mutants was measured at saturating Na(+) concentration. In contrast, the other type of mutations (A127V and A127T) was found to affect both the K(m) and the pH dependence of the rate of NhaA whether tested at saturating Na(+) concentration or not. These results imply that residues involved in the ion translocation of NhaA may (A127) or may not (D133, T132, and P129) overlap with those affecting the pH response of the antiporter. All mutants cluster in the N-terminal half of the putative alpha-helix IV, one type on one face, the other on the opposite. Cys accessibility test demonstrated that although D133C is located in the middle of TMS IV, it is inhibited by N-ethylmaleimide and is exposed to the cytoplasm.

The crystal structure of Escherichia coli NhaA determined at pH 4 has provided insights into the mechanism of activity of a pH-regulated Na ؉ /H ؉ antiporter. However, becauseNhaA is activated at physiological pH (pH 5.5-8.5), many questions related to the active state of NhaA have remained elusive. Our experimental results at physiological pH and computational analyses reveal that amino acid residues in transmembrane segment II contribute to the cation pathway of NhaA and its pH regulation: 1) transmembrane segment II is a highly conserved helix and the conserved amino acid residues are located on one side of the helix facing either the cytoplasmic or periplasmic funnels of NhaA structure. 2) Cys replacements of the conserved residues and measuring their antiporter activity in everted membrane vesicles showed that D65C, L67C, E78C, and E82C increased the apparent K m to Na ؉ and Li Sodium proton antiporters are ubiquitous membrane proteins found in the cytoplasmic and organelle membranes of cells of many different origins, including plants, animals, and microorganisms. They are involved in cell energetics and play primary roles in the regulation of intracellular pH, cellular Na ϩ content, and cell volume (see reviews in Refs. 1-3).

The 3D structure of Escherichia coli NhaA, determined at pH 4, provided the first structural insights into the mechanism of antiport and pH regulation of a Na+/H+ antiporter. However, because NhaA is activated at physiological pH (pH 7.0-8.5), many questions pertaining to the active state of NhaA have remained open, including the physiological role of helix X. Using a structural-based evolutionary approach in silico, we identified a segment of most conserved residues in the middle of helix X. These residues were then used as targets for functional studies at physiological pH. Cysteine-scanning mutagenesis showed that Gly303, in the middle of the conserved segment, is an essential residue and Cys replacement of Lys300 retains only Li+/H+ antiporter activity, with a 20-fold increase in the apparent KM for Li+. Cys replacements of Leu296 and Gly299 increase the apparent KM of the Na+/H+ antiporter for both Na+ and Li+. Accessibility test to N-ethylmaleimide and 2-sulfonatoethyl methanethiosulfonate showed that G299C, K300C, and G303C are accessible to the cytoplasm. Suppressor mutations and site-directed chemical cross-linking identified a functional and/or structural interaction between helix X (G295C) and helix IVp (A130C). While these results were in accordance with the acid-locked crystal structure, surprisingly, conflicting data were also obtained; E78C of helix II cross-links very efficiently with several Cys replacements of helix X, and E78K/K300E is a suppressor mutation of K300E. These results reveal that, at alkaline pH, the distance between the conserved center of helix X and E78 of helix II is drastically decreased, implying a pH-induced conformational change of one or both helices.