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 three-dimensional crystal 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 6.5-8.5), many questions pertaining to the active state of NhaA have remained open including the structural and physiological roles of helix IX and its loop VIII-IX. Here we studied this NhaA segment (Glu 241 -Phe 267 ) by structure-based biochemical approaches at physiological pH. Cysteine-scanning mutagenesis identified new mutations affecting the pH dependence of NhaA, suggesting their contribution to the "pH sensor." Furthermore mutation F267C reduced the H ؉ /Na ؉ stoichiometry of the antiporter, and F267C/F344C inactivated the antiporter activity. Tests of accessibility to [2-(trimethylammonium)ethyl]methanethiosulfonate bromide, a membrane-impermeant positively charged SH reagent with a width similar to the diameter of hydrated Na ؉ , suggested that at physiological pH the cytoplasmic cation funnel is more accessible than at acidic pH. Assaying intermolecular cross-linking in situ between single Cys replacement mutants uncovered the NhaA dimer interface at the cytoplasmic side of the membrane; between Leu 255 and the cytoplasm, many Cys replacements cross-link with various cross-linkers spanning different distances (10 -18 Å ) implying a flexible interface. L255C formed intermolecular S-S bonds, cross-linked only with a 5-Å cross-linker, and when chemically modified caused an alkaline shift of 1 pH unit in the pH dependence of NhaA and a 6-fold increase in the apparent K m for Na ؉ of the exchange activity suggesting a rigid point in the dimer interface critical for NhaA activity and pH regulation.Regulation of intracellular pH, cellular Na ϩ content, and cell volume is essential for all living cells. Na ϩ /H ϩ antiporters play primary roles in these crucial processes. They are integral membrane proteins, ubiquitous throughout the biological kingdom. Many Na ϩ /H ϩ antiporters are tightly regulated by pH, a property that underpins their capacity to maintain pH homeostasis of the cytoplasm (1).NhaA, the main Na ϩ /H ϩ antiporter of Escherichia coli, has eukaryotic orthologs, including human (2, 3). It is an electrogenic antiporter with a stoichiometry of 2 H ϩ /1 Na ϩ (1, 4) and is strongly dependent on pH; its rate of activity changes over 3 orders of magnitude between pH 7.0 and 8.5 (1, 4, 5).NhaA is a dimer in the native membrane as revealed by genetic complementation data, biochemical pulldown experiments, intermolecular cross-linking (6), ESR studies (7,8), and cryoelectron microscopy of two-dimensional crystals (9, 10). The recently determined crystal structure of NhaA monomer at pH 4 (11) has provided the first structural insights into the mechanism of antiport and pH regulation of a Na ϩ /H ϩ antiporter. NhaA consists of 12 TMSs 2 with the N and C termini pointing into the cytoplasm. It represents a novel fold; TMSs IV and XI are each comprised ...

NhaA, the Na ؉ /H ؉ antiporter of Escherichia coli, exists in the native membrane as a homodimer of which two monomers have been suggested to be attached by a ␤-hairpin at the periplasmic side of the membrane. Constructing a mutant deleted of the ␤-hairpin, NhaA/⌬(Pro 45 -Asn 58 ), revealed that in contrast to the dimeric mobility of native NhaA, the mutant has the mobility of a monomer in a blue native gel. Intermolecular cross-linking that monitors dimers showed that the mutant exists only as monomers in the native membrane, proteoliposomes, and when purified in ␤-dodecyl maltoside micelles. Furthermore, pulldown experiments revealed that, whereas as expected for a dimer, hemagglutinin-tagged wild-type NhaA co-purified with His-tagged NhaA on a Ni 2؉ -NTA affinity column, a similar version of the mutant did not. Remarkably, under routine stress conditions (0.1 M LiCl, pH 7 or 0.6 M NaCl, pH 8.3), the monomeric form of NhaA is fully functional. It conferred salt resistance to NhaA-and NhaB-deleted cells, and whether in isolated membrane vesicles or reconstituted into proteoliposomes exhibited Na ؉ /H ؉ antiporter activity and pH regulation very similar to wild-type dimers. Remarkably, under extreme stress conditions (0.1 M LiCl or 0.7 M NaCl at pH 8.5), the dimeric native NhaA was much more efficient than the monomeric mutant in conferring extreme stress resistance.

The unique trypsin cleavable site of NhaA, the Na(+)/H(+) antiporter of Escherichia coli, was exploited to detect a change in mobility of cross-linked products of NhaA by polyacrylamide gel electrophoresis. Double-Cys replacements were introduced into loops, one on each side of the trypsin cleavage site (Lys 249). The proximity of paired Cys residues was assessed by disulfide cross-linking of the two tryptic fragments, using three homobifunctional cross-linking agents: 1,6-bis(maleimido)hexane (BMH), N,N'-o-phenylenedimaleimide (o-PDM), and N,N'-p-phenylenedimaleimide (p-PDM). The interloop cross-linking was found to be very specific, indicating that the loops are not merely random coils that interact randomly. In the periplasmic side of NhaA, two patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently between the double-Cys replacements A118C/S286C, N177C/S352C, and H225C/S352C; (b) only BMH cross-links the double-Cys replacements A118C/S352C, N177C/S286C, and H225C/S286C. In the cytoplasmic side of NhaA, three patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently the pairs of Cys replacements L4C/E252C, S146C/L316C, S146C/R383C, and E241C/E252C; (b) BMH and p-PDM cross-link efficiently the pairs of Cys replacements S87C/E252C, S87C/L316C, and S146C/E252C; (c) none of the reagents cross-links the double-Cys replacements L4C/L316C, L4C/R383C, S87C/R383C, A202C/E252C, A202C/L316C, A202C/R383C, E241C/L316C, and E241C/R383C. The data reveal that the N-terminus and loop VIII-IX that have previously been shown to change conformation with pH are in close proximity within the NhaA protein. The data also suggest close proximity between N-terminal and C-terminal helices at both the cytoplasmic and the periplasmic face of NhaA.

A single Cys replacement of Glu at position 252 (E252C) in loop VIII-IX of NhaA increases drastically the K m for Na ؉ (50-fold) of the Na ؉ /H ؉ antiporter activity of NhaA and shifts the pH dependence of NhaA activity, by one pH unit, to the alkaline range. In parallel, E252C causes a similar alkaline pH shift to the pH-induced conformational change of loop VIII-IX. Thus, although both the Na ؉ /H ؉ antiporter activity of wild type NhaA and its accessibility to trypsin at position Lys 249 in loop VIII-IX increase with pH between pH 6.5 and 7.5, the response of E252C occurs above pH 8. Furthermore, probing accessibility of pure E252C protein in dodecyl maltoside solution to 2-(4-maleimidylanilino)-naphthalene-6-sulfonic acid revealed that E252C itself undergoes a pH-dependent conformational change, similar to position Lys 249 , and the rate of the pH-induced conformational change is increased specifically by the presence of Na ؉ or Li ؉ , the specific ligands of the antiporter. Chemical modification of E252C by N-ethylmaleimide, 2-(4-maleimidylanilino)-naphthalene-6-sulfonic acid; [2-(trimethylammonium)ethyl]methane thiosulfonate, or (2-sulfonatoethyl)methanethiosulfonate reversed, to a great extent, the pH shift conferred by E252C but had no effect on the K m of the mutant antiporter.