Structure-function relationship of the fifth transmembrane domain in the Na+/H+ antiporter of Helicobacter pylori: Topology and function of the residues, including two consecutive essential aspartate residues

We examined the structure-function relationships of residues in the fifth transmembrane domain (TM5) of the Na+/H+ antiporter A (NhaA) from Helicobacter pylori (HP NhaA) by cysteine scanning mutagenesis. TM5 contains two aspartate residues, Asp-171 and Asp-172, which are essential for antiporter activity. Thirty-five residues spanning the putative TM5 and adjacent loop regions were replaced by cysteines. Cysteines replacing Val-162, Ile-165, and Asp-172 were labeled with NEM, suggesting that these three residues are exposed to a hydrophilic cavity within the membrane. Other residues in the putative TM domain, including Asp-171, were not labeled. Inhibition of NEM labeling by the membrane impermeable reagent AMS suggests that Val-162 and Ile-165 are exposed to a water filled channel open to the cytoplasmic space, whereas Asp-172 is exposed to the periplasmic space. D171C and D172C mutants completely lost Na+/H+ and Li+/H+ antiporter activities, whereas other Cys replacements did not result in a significant loss of these activities. These results suggest that Asp-171 and Asp-172 and the surrounding residues of TM5 provide an essential structure for H+ binding and Na+ or Li+ exchange. A168C and Y183C showed markedly decreased antiporter activities at acidic pH, whereas their activities were higher at alkaline pH, suggesting that the conformation of TM5 also plays a crucial role in the HP NhaA-specific acidic pH antiporter activity.     

Identification of membrane domains of the Na+/H+ antiporter (NhaA) protein from Helicobacter pylori required for ion transport and pH sensing

The Na+/H+ antiporter from Helicobacter pylori (HP NhaA) is normally active within the pH range 6.0-8.5. In contrast, the NhaA from Escherichia coli (EC NhaA) is active only within the alkaline pH range 7.5-8.5. We studied structures of HP NhaA involved in ion transport and pH sensing by analyzing mutants with defects in NhaA activity. The 36 mutants were classified into three types. The first type exhibited very low or null activity at all pH levels and had amino acid substitutions in the transmembrane segments (TM) 4, 5, 10, and 11, implicating these TMs in ion transport. The second type, which had amino acid substitutions at Met-138, Phe-144, and Lys-347 in TM 4 and 10, exhibited very low antiporter activity at acidic pH but had significantly higher activity at alkaline pH. These results imply that TM 4 (Met-138 and Phe-144) and 10 (Lys-347) are involved in supporting transport activity at acidic pH, in addition to their essential role in the overall transport mechanism. The third type of mutant exhibited very low antiporter activity at alkaline pH but relatively normal activity at acidic pH and had amino acid substitutions in loop 7 (a hydrophilic region between TM 7 and 8) as well as in TM 8, suggesting that these regions are involved in antiporter activation at alkaline pH. Three revertants that suppress a Lys-347 mutation were identified. Two of three suppressor mutations were located in loops 2 and 4, suggesting a functional interaction between these regions (loops 2 and 4 and TM 10). Thus, HP NhaA activity may be modulated by two independent factors that are dependent on pH: an activation mechanism at acidic pH, which is regulated by residues within TM 4 and 10 and another mechanism functioning at alkaline pH regulated by residues within loop 7 and TM 8.     

Cysteine-scanning mutagenesis of transmembrane segments 4 and 5 of the Tn10-encoded metal-tetracycline/H+ antiporter reveals a permeability barrier in the middle of a transmembrane water-filled channel

Cysteine-scanning mutants as to putative transmembrane segments 4 and 5 and the flanking regions of Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) were constructed. All mutants were normally expressed. Among the 57 mutants (L99C to I155C), nine conserved arginine-, aspartate-, and glycine-replaced ones exhibited greatly reduced tetracycline resistance and almost no transport activity, and five conserved glycine- and proline-replaced mutants exhibited greatly reduced tetracycline transport activity in inverted membrane vesicles despite their high or moderate drug resistance. All other cysteine-scanning mutants retained normal drug resistance and normal tetracycline transport activity except for the L142C and I143C mutants. The transmembrane (TM) regions TM4 and TM5 were determined to comprise 20 amino acid residues, Leu-99 to Ile-118, and 17 amino acid residues, Ala-136 to Ala-152, respectively, on the basis of N-[(14)C]ethylmaleimide ([(14)C]NEM) reactivity. The NEM reactivity patterns of the TM4 and TM5 mutants were quite different from each other. TM4 could be divided into two halves, that is, a NEM nonreactive periplasmic half and a periodically reactive cytoplasmic half, indicating that TM4 is tilted toward a water-filled transmembrane channel and that only its cytoplasmic half faces the channel. On the other hand, NEM-reactive mutations were observed periodically (every two residues) along the whole length of TM5. A permeability barrier for a membrane-impermeable sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, was present in the middle of TM5 between Leu-142 and Gly-145, whereas all the NEM-reactive mutants as to TM4 were not accessible to 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, indicating that the channel-facing side of TM4 is located inside the permeability barrier. Tetracycline protected the G141C mutant from the NEM binding, whereas the other mutants in TM4 and TM5 were not protected by tetracycline.     

Functional and structural interactions of the transmembrane domain X of NhaA, Na+/H+ antiporter of Escherichia coli, at physiological pH

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.     

Unraveling functional and structural interactions between transmembrane domains IV and XI of NhaA Na+/H+ antiporter of Escherichia coli

A functionally important, interface domain between transmembrane segments (TMSs) IV and XI of the NhaA Na+/H+ antiporter of Escherichia coli has been unraveled. Scanning by single Cys replacements identified new mutations (F136C, G125C, and A137C) that cluster in one face of TMS IV and increase dramatically the Km of the antiporter. Whereas G125C, in addition, causes a drastic alkaline shift to the pH dependence of the antiporter, G338C alleviates the pH control of NhaA. Scanning by double Cys replacements (21 pairs of one replacement per TMS) identified genetically eight pairs of residues that showed very strong negative complementation. Cross-linking of the double mutants identified six double mutants (T132C/G338C, D133C/G338C, F136C/S342C, T132C/S342C, A137C/S342C, and A137C/G338C) of which pronounced intramolecular cross-linking defined an interface domain between the two TMSs. Remarkably, cross-linking by a short and rigid reagent (N,N'-o-phenylenedimaleimide) revived the Li+/H+ antiport activity, whereas a shorter reagent (1,2-ethanediyl bismethanethiosulfonate) revived both Na+/H+ and Li+/H+ antiporter activities and even the pH response of the dead mutant T132C/G338C. Hence, cross-linking at this position restores an active conformation of NhaA.     

Cysteine-scanning mutagenesis around transmembrane segments 1 and 11 and their flanking loop regions of Tn10-encoded metal-Tetracycline/H+ antiporter

Putative transmembrane helices (TM) 1 and 11 in the metal-tetracycline/H(+) antiporter are predicted to be close to each other on the basis of disulfide cross-linking experiments of the double-cysteine mutants in the periplasmic loop regions (Kubo, Y., Konishi, S., Kawabe, T., Nada, S., and Yamaguchi, A. (2000) J. Biol. Chem. 275, 5270-5274). In this study, each amino acid from Asn-2 to Gly-44 in the putative TM1 and loop1-2 regions or that from Ser-328 to Gly-366 in TM11 and its flanking regions was individually replaced with cysteine. With respect to the TM1 region, 10 mutants, from T5C to L14C, were all not reactive with N-ethylmaleimide (NEM), and from D15C to I22C, NEM-reactive and non-reactive mutations periodically appeared every two residues. Three mutants, M23C to V25C, were all NEM-reactive, but the degree of the latter two mutants was very low. Seven mutants, from L26C to E32C, were all highly reactive with NEM. Therefore, the region of TM1 is composed of the 21 amino acid residues from Thr-5 to Val-25. It is a partially amphiphilic helix, that is, the N-terminal (cytoplasmic) half is embedded in the hydrophobic interior, and the C-terminal (periplasmic) half faces a water-filled channel. With respect to TM11, nine mutants, from S328C to G336C, and six mutants, from L361C to G366C, were all reactive with NEM. On the other hand, out of the 24 mutants, from L337C to S360C, 17 were not reactive with NEM, and the 7 NEM-reactive mutants were scattered, indicating that this region is a transmembrane segment. The 7 residues from Val-347 to Phe-353 including Pro-350 formed a central hydrophobic core, and the 7 NEM-reactive mutations were periodically distributed in its flanking regions, indicating that both ends of TM11 face a water-filled channel. Ala-354 is located at about 1/3 of the length from the periplasmic end of TM11. Disulfide cross-linking experiments on double-cysteine mutants having the combination of A354C and a cysteine-scanning mutation in the loop1-2 region indicated that loop1-2 is very flexible and close to the periplasmic end of TM11. Tetracycline prevented the cross-linking formation between the periplasmic ends of TM1 and TM11; however, it did not affect the cross-linking between loop1-2 and TM11, indicating that the substrate-induced conformational change involves a shift in the relative locations of TM1 and TM11.     

Roles of NhaA,NhaB, and NhaD Na+/H+ antiporters in survival of Vibrio cholerae in a saline environment

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.     

Kinetics of charge translocation in the passive downhill uptake mode of the Na+/H+ antiporter NhaA of Escherichia coli

The Na+/H+ antiporter NhaA is the main Na+ extrusion system in E. coli. Using direct current measurements combined with a solid supported membrane (SSM), we obtained electrical data of the function of NhaA purified and reconstituted in liposomes. These measurements demonstrate NhaA's electrogenicity, its specificity for Li+ and Na+ and its pronounced pH dependence in the range pH 6.5-8.5. The mutant G338S, in contrast, presents a pH independent profile, as reported previously. A complete right-side-out orientation of the NhaA antiporter within the proteoliposomal membrane was determined using a NhaA-specific antibody based ELISA assay. This allowed for the first time the investigation of NhaA in the passive downhill uptake mode corresponding to the transport of Na+ from the periplasmic to the cytoplasmic side of the membrane. In this mode, the transporter has kinetic properties differing significantly from those of the previously investigated efflux mode. The apparent Km values were 11 mM for Na+ and 7.3 mM for Li+ at basic pH and 180 mM for Na+ and 50 mM for Li+ at neutral pH. The data demonstrate that in the passive downhill uptake mode pH regulation of the carrier affects both apparent Km as well as turnover (Vmax).     

Mutational analysis of amiloride sensitivity of the NhaA Na+/H+ antiporter from Vibrio parahaemolyticus.

The activity of the NhaA Na+/H+ antiporter of Vibrio parahaemolyticus is inhibited by amiloride. We found an amino acid sequence in the NhaA that was identical to a putative amiloride binding domain of the Na+/H+ exchanger in mammalian cells. We constructed mutant NhaAs that had amino acid substitutions in the putative amiloride binding domain by site-directed mutagenesis. These include V62L (Val62 replaced by Leu), F63Y, F64Y, and L65F. Most mutant NhaAs showed decreased sensitivity for amiloride. Among these, the F64Y mutant NhaA showed the least amiloride sensitivity, with a Ki value 7 to 10 times greater than that in the wild type. Thus, the sequence between residues V62 and L65 in NhaA, especially F64, is very important for the inhibitory effect of amiloride on the antiporter.     

Trans membrane domain IV is involved in ion transport activity and pH regulation of the NhaA-Na(+)/H(+) antiporter of Escherichia coli.

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 Na+/H+ antiporter of the thermohalophilic bacterium Rhodothermus marinus

In the thermohalophilic bacterium Rhodothermus marinus, the NADH:quinone oxidoreductase (complex I) is encoded by two single genes and two operons, one of which contains the genes for five complex I subunits, nqo10-nqo14, a pterin carbinolamine dehydratase, and a putative single subunit Na+/H+ antiporter. Here we report that the latter encodes indeed a functional Na+/H+ antiporter, which is able to confer resistance to Na+, but not to Li+ to an Escherichia coli strain defective in Na+/H+ antiporters. In addition, an extensive amino acid sequence comparison with several single subunit Na+/H+ antiporters from different groups, namely NhaA, NhaB, NhaC, and NhaD, suggests that this might be the first member of a new type of Na+/H+ antiporters, which we propose to call NhaE.     

Oligomerization of NhaA, the Na+/H+ antiporter of Escherichia coli in the membrane and its functional and structural consequences

Recently, a two-dimensional crystal structure of NhaA, the Na+/H+ antiporter of Escherichia coli has been obtained [Williams, K. A., Kaufer, U. G., Padan, E., Schuldiner, S. and Kühlbrandt, W. (1999) EMBO J., 18, 3558-3563]. In these crystals NhaA exists as a dimer. Using biochemical and genetic approaches here we show that NhaA exists in the native membrane as a homooligomer. Functional complementation between the polypeptides of NhaA was demonstrated by coexpression of pairs of conditional lethal (at high pH in the presence of Na+) mutant alleles of nhaA in EP432, a strain lacking antiporters. Physical interaction in the membrane was shown between the His-tagged NhaA polypeptide which is readily affinity purified from DM-solubilized membranes with a Ni2+-NTA column and another which is not; only when coexpressed did both copurify on the column. The organization of the oligomer in the membrane was studied in situ by site-directed cross-linking experiments. Cysteine residues were introduced--one per NhaA--into certain loops of Cys-less NhaA, so that only intermolecular cross-linking could take place. Different linker-size cross-linkers were applied to the membranes, and the amount of the cross-linked protein was analyzed by mobility shift on SDS-PAGE. The results are consistent with homooligomeric NhaA and the location of residue 254 in the interface between monomers. Intermolecular cross-linking of V254C caused an acidic shift in the pH profile of NhaA.     

Functional characterization of a NapA Na(+)/H(+) antiporter from Thermus thermophilus

Na(+)/H(+) antiporters are ubiquitous membrane proteins and play an important role in cell homeostasis. We amplified a gene encoding a member of the monovalent cation:proton antiporter-2 (CPA2) family (TC 2.A.37) from the Thermus thermophilus genome and expressed it in Escherichia coli. The gene product was identified as a member of the NapA subfamily and was found to be an active Na(+)(Li(+))/H(+) antiporter as it conferred resistance to the Na(+) and Li(+) sensitive strain E. coli EP432 (DeltanhaA, DeltanhaB) upon exposure to high concentration of these salts in the growth medium. Fluorescence measurements using the pH sensitive dye 9-amino-6-chloro-2-methoxyacridine in everted membrane vesicles of complemented E. coli EP432 showed high Li(+)/H(+) exchange activity at pH 6, but marginal Na(+)/H(+) antiport activity. Towards more alkaline conditions, Na(+)/H(+) exchange activity increased to a relative maximum at pH 8, where by contrast the Li(+)/H(+) exchange activity reached its relative minimum. Substitution of conserved residues D156 and D157 (located in the putative transmembrane helix 6) with Ala resulted in the complete loss of Na(+)/H(+) activity. Mutation of K305 (putative transmembrane helix 10) to Ala resulted in a compromised phenotype characterized by an increase in apparent K(m) for Na(+) (36 vs. 7.6 mM for the wildtype) and Li(+) (17 vs. 0.22 mM), In summary, the Na(+)/H(+) antiport activity profile of the NapA type transporter of T. thermophilus resembles that of NhaA from E. coli, whereas in contrast to NhaA the T. thermophilus NapA antiporter is characterized by high Li(+)/H(+) antiport activity at acidic pH.     

The putative Na+/H+ antiporter (NapA) of Enterococcus hirae is homologous to the putative K+/H+ antiporter (KefC) of Escherichia coli

Two monovalent ion porters, the putative Na+/H+ antiporter (NapA) of Enterococcus hirae and the putative K+/H+ antiporter (KefC) of Escherichia coli, are similar in sequence throughout their hydrophobic domains. These two proteins, which comprise a novel family of transporters unrelated to the previously characterized Na+/H+ exchangers of E. coli (NhaA and NhaB) are proposed to function by essentially the same mechanism.     

Mutation E252C increases drastically the Km value for Na+ and causes an alkaline shift of the pH dependence of NhaA Na+/H+ antiporter of Escherichia coli

A single Cys replacement of Glu at position 252 (E252C) in loop VIII-IX of NhaA increases drastically the Km 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.     

Amino acid substitutions and alteration in cation specificity in the melibiose carrier of Escherichia coli

We isolated mutants of Escherichia coli which showed Li+-resistant growth on melibiose. The melibiose carrier of the mutants lost the ability to couple to H+, whereas it retained the ability to couple to Na+. The mutated gene, melB, of the mutants was cloned, and the nucleotide sequence was determined. The nucleotide replacements caused the following substitutions of amino acid residues in the melibiose carrier: Pro-142 with Ser, Leu-232 with Phe, or Ala-236 with Thr or Val. These amino acid residues are located in slightly hydrophobic regions of the melibiose carrier. The results provide strong support for the idea that such regions or their vicinities which contain those amino acid residues play an important role in H+ (or Li+) recognition or H+ (or Li+) transport by the melibiose carrier.     

Detection of oligomerization and conformational changes in the Na+/H+ antiporter from Helicobacter pylori by fluorescence resonance energy transfer

Oligomerization and conformational changes in the Na+/H+ antiporter from Helicobacter pylori (HPNhaA) were studied by means of fluorescence resonance energy transfer (FRET) analysis. Na+/H+ antiporter-deficient Escherichia coli cells expressing C-terminal fusions of HPNhaA to green fluorescent protein (GFP) variants exhibited wild-type levels of antiporter activity in their everted membrane vesicles. Vesicles containing both HPNhaA-CFP and HPNhaA-YFP or HPNhaA-Venus exhibited FRET from CFP (donor) to YFP or Venus (acceptor), suggesting that HPNhaA forms an oligomer. Co-precipitation of HPNhaA tagged by Venus and FLAG sequences confirmed oligomerization. FRET decreased extensively after treatment of the vesicles with proteinase K, which released GFP variants from the fusion proteins. FRET was not observed by merely mixing vesicles expressing the donor or acceptor fusion alone. Fluorescence of Venus is less sensitive to anions and stronger than that of anion-sensitive YFP. Using HPNhaA-Venus as the acceptor, Li+ was found to cause a significant decrease in FRET regardless of the presence or absence of DeltapH across the membranes, whereas Na+ caused a much weaker effect. This Li+ effect was minimal in vesicles prepared from cells expressing HPNhaA containing an Asp141 to Asn mutation, which results in defective Li+/H+ antiporter activity, possibly Li+ binding. These results demonstrate that monomer interactions within the HPNhaA oligomer are weakened possibly by Li+ binding. Dynamic interactions between HPNhaA monomers were detectable in membranes by FRET analysis, thus providing a new approach to study dynamic conformational changes in NhaA during antiport activity.     

Transmembrane Segment II of NhaA Na+/H+ Antiporter Lines the Cation Passage, and Asp65 Is Critical for pH Activation of the Antiporter

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, because NhaA 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⁺ and changed the pH response of the antiporter. 3) Introduced Cys replacements, L60C, N64C, F71C, F72C, and E78C, were significantly alkylated by [¹⁴C]N-ethylmaleimide implying the presence of water-filled cavities in NhaA. 4) Several Cys replacements were modified by MTSES and/or MTSET, membrane impermeant, negatively and positively charged reagents, respectively, that could reach Cys replacements from the periplasm only via water-filled funnel(s). Remarkably, the reactivity of D65C to MTSES increased with increasing pH and chemical modification by MTSES but not by MTSET, decreased the apparent K_m of the antiporter at pH 7.5 (10-fold) but not at pH 8.5, implying the importance of Asp⁶⁵ negative charge for pH activation of the antiporter.     

Chimeric Na(+)/H(+) antiporters constructed from NhaA of Helicobacter pylori and Escherichia coli: implications for domains of NhaA for pH sensing

In order to delineate regions which play a role in the regulation of Na(+)/H(+) antiporter NhaA activity by pH, we analyzed the antiporter activities of various chimeric mutants constructed from specific portions of NhaA from Escherichia coli and Helicobacter pylori (EC and HP NhaA). HP NhaA contains 10 residues at the amino-terminus, and 38 residues in a loop region between the eighth and ninth transmembrane spans (loop 8), which are absent in EC NhaA. Deletion from HP NhaA or insertion into EC NhaA of the sequences caused almost no change in pH-dependent antiport activities relative to in the case of the wild-type parent molecules. Chimeras consisting of various combinations of the amino-terminal (amino terminus to sixth or eighth transmembrane span) and carboxy-terminal (seventh or ninth transmembrane span to the carboxy-terminus) regions of EC and HP NhaA showed antiporter activity profiles intermediate between those of the parent molecules. These results show that the two HP-specific sequences are not directly involved in the mechanism of pH sensing by HP NhaA and that the pH sensitivity of NhaA activity is not determined by the amino- or carboxy-terminal regions of NhaA alone, but may be due to interaction between unconserved residues in the two domains. In addition, it was suggested that loop 8 functions primarily as a hinge in both NhaA molecules.     

Importance of the seryl and threonyl residues of the fifth transmembrane domain to the substrate specificity of yeast plasma membrane Na+/H+ antiporters

The Zygosaccharomyces rouxii Na+/H+ antiporter Sod2-22p is a member of the subfamily of yeast plasma membrane Nha/Sod antiporters that do not recognize potassium as their substrate. A functional study of two ZrSod2-22p mutated versions that improved the tolerance of a S. cerevisiae alkali-metal-cation sensitive strain to high extracellular concentration of KCl identified two polar non-charged amino-acid residues in the fifth transmembrane domain, Thr141 and Ser150, as being involved in substrate recognition and transport in yeast Nha/Sod antiporters. A reciprocal substitution of amino-acid residues with a hydroxyl group at these positions, T141S or S150T, produced a broadened cation selectivity of the antiporter for K+, in addition to Na+ and Li+. Site-directed mutagenesis of Ser150 showed that while the replacement of Ser150 with a small hydrophobic (valine) or negatively charged (aspartate) amino acid did not produce a significant change in ZrSod2-22p substrate specificity, the introduction of a positive charge at this position stopped the activity of the antiporter. This data demonstrates that the amino-acid composition of the fifth transmembrane domain, mainly the presence of amino acids containing hydroxyl groups in this part of the protein, is critical for the recognition and transport of substrates and could participate in conformational movements during the binding and/or cation transport cycle in yeast plasma membrane Na+/H+ antiporters.