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.     

Structure-based functional study reveals multiple roles of transmembrane segment IX and loop VIII-IX in NhaA Na+/H+ antiporter of Escherichia coli at physiological pH

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 A) implying a flexible interface. L255C formed intermolecular S-S bonds, cross-linked only with a 5-A 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.     

A pH-dependent conformational change of NhaA Na(+)/H(+) antiporter of Escherichia coli involves loop VIII-IX, plays a role in the pH response of the protein, and is maintained by the pure protein in dodecyl maltoside.

Digestion with trypsin of purified His-tagged NhaA in a solution of dodecyl maltoside yields two fragments at alkaline pH but only one fragment at acidic pH. Determination of the amino acid sequence of the N terminus of the cleavage products show that the pH-sensitive cleavage site of NhaA, both in isolated everted membrane vesicles as well as in the pure protein in detergent, is Lys-249 in loop VIII-IX, which connects transmembrane segment VIII to IX. Interestingly, the two polypeptide products of the split antiporter remain complexed and co-purify on Ni(2+)-NTA column. Loop VIII-IX has also been found to play a role in the pH regulation of NhaA; three mutations introduced into the loop shift the pH profile of the Na(+)/H(+) antiporter activity as measured in everted membrane vesicles. An insertion mutation introducing Ile-Glu-Gly between residues Lys-249 and Arg-250 (K249-IEG-R250) and Cys replacement of either Val-254 (V254C) or Glu-241 (E241C) cause acidic shift of the pH profile of the antiporter by 0.5, 1, and 0.3 pH units, respectively. Interestingly, the double mutant E241C/V254C introduces a basic shift of more than 1 pH unit with respect to the single mutation V254C. Taken together these results imply the involvement of loop VIII-IX in the pH-induced conformational change, which leads to activation of NhaA at alkaline pH.     

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.     

Transport mechanism and pH regulation of the Na+/H+ antiporter NhaA from Escherichia coli: an electrophysiological study

Using an electrophysiological assay the activity of NhaA was tested in a wide pH range from pH 5.0 to 9.5. Forward and reverse transport directions were investigated at zero membrane potential using preparations with inside-out and right side-out-oriented transporters with Na(+) or H(+) gradients as the driving force. Under symmetrical pH conditions with a Na(+) gradient for activation, both the wt and the pH-shifted G338S variant exhibit highly symmetrical transport activity with bell-shaped pH dependences, but the optimal pH was shifted 1.8 pH units to the acidic range in the variant. In both strains the pH dependence was associated with a systematic increase of the K(m) for Na(+) at acidic pH. Under symmetrical Na(+) concentration with a pH gradient for NhaA activation, an unexpected novel characteristic of the antiporter was revealed; rather than being down-regulated, it remained active even at pH as low as 5. These data allowed a transport mechanism to advance based on competing Na(+) and H(+) binding to a common transport site and a kinetic model to develop quantitatively explaining the experimental results. In support of these results, both alkaline pH and Na(+) induced the conformational change of NhaA associated with NhaA cation translocation as demonstrated here by trypsin digestion. Furthermore, Na(+) translocation was found to be associated with the displacement of a negative charge. In conclusion, the electrophysiological assay allows the revelation of the mechanism of NhaA antiport and sheds new light on the concept of NhaA pH regulation.     

The influence of protonation states on the dynamics of the NhaA antiporter from Escherichia coli

The crystal structure of NhaA Na(+)/H(+) antiporter of Escherichia coli has provided a basis to explore the mechanism of Na(+) and H(+) exchange and its regulation by pH. However, the dynamics and nature of the pH-induced changes in the proteins remained unknown. Using molecular mechanics methods, we studied the dynamic behavior of the hydrogen-bonded network in NhaA on shifting the pH from 4 to 8. The helical regions preserved the general architecture of NhaA throughout the pH change. In contrast, large conformational drifts occurred at pH 8 in the loop regions, and an increased flexibility of helix IVp was observed on the pH shift. A remarkable pH-induced conformational reorganization was found: at acidic pH helix X is slightly curved, whereas at alkaline pH, it is kinked around residue Lys(300). The barrier that exists between the cytoplasmic and periplasmic funnels at low pH is removed, and the two funnels are bridged by hydrogen bonds between water molecules and residues located in the TMSs IV/XI assembly and helix X at alkaline pH. In the variant Gly(338)Ser that lost pH control, a hydrogen-bonded chain between Ser(338) and Lys(300) was found to block the pH-induced conformational reorganization of helix X.     

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.     

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.     

NhaA of Escherichia coli, as a model of a pH-regulated Na+/H+antiporter

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.     

Assessing oligomerization of membrane proteins by four-pulse DEER: pH-dependent dimerization of NhaA Na+/H+ antiporter of E. coli

The pH dependence of the structure of the main Na(+)/H(+) antiporter NhaA of Escherichia coli is studied by continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) techniques on singly spin-labeled mutants. Residues 225 and 254 were selected for site-directed spin labeling, as previous work suggested that they are situated in domains undergoing pH-dependent structural changes. A well-defined distance of 4.4 nm between residues H225R1 in neighboring molecules is detected by a modulation in double electron-electron resonance data. This indicates that NhaA exists as a dimer, as previously suggested by a low-resolution electron density map and cross-linking experiments. The modulation depth decreases reversibly when pH is decreased from 8 to 5.8. A quantitative analysis suggests a dimerization equilibrium, which depends moderately on pH. Furthermore, the mobility and polarity of the environment of a spin label attached to residue 225 change only slightly with changing pH, while no other changes are detected by CW EPR. As antiporter activity of NhaA changes drastically in the studied pH range, residues 225 and 254 are probably located not in the sensor or ion translocation sites themselves but in domains that convey the signal from the pH sensor to the translocation site.     

Epitope mapping of conformational monoclonal antibodies specific to NhaA Na+/H+ antiporter: structural and functional implications

The recently determined crystal structure of NhaA, the Na ⁺/H ⁺ antiporter of Escherichia coli, showed that the previously constructed series of NhaA-alkaline phosphatase (PhoA) fusions correctly predicted the topology of NhaA's 12 transmembrane segments (TMS), with the C- and N-termini pointing to the cytoplasm. Here, we show that these NhaA-PhoA fusions provide an excellent tool for mapping the epitopes of three NhaA-specific conformational monoclonal antibodies (mAbs), of which two drastically inhibit the antiporter. By identifying which of the NhaA fusions is bound by the respective mAb, the epitopes were localized to small stretches of NhaA. Then precise mapping was conducted by targeted Cys scanning mutagenesis combined with chemical modifications. Most interestingly, the epitopes of the inhibitory mAbs, 5H4 and 2C5, were identified in loop X-XI (cytoplasmic) and loop XI-XII (periplasmic), which are connected by TMS XI on the cytoplasmic and periplasmic sides of the membrane, respectively. The revealed location of the mAbs suggests that mAb binding distorts the unique NhaA TMS IV/XI assembly and thus inhibits the activity of NhaA. The noninhibitory mAb 6F9 binds to the functionally dispensable C-terminus of NhaA.     

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.     

Monomers of the NhaA Na+/H+ antiporter of Escherichia coli are fully functional yet dimers are beneficial under extreme stress conditions at alkaline pH in the presence of Na+ or Li+

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 beta-hairpin at the periplasmic side of the membrane. Constructing a mutant deleted of the beta-hairpin, NhaA/Delta(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 beta-dodecyl maltoside micelles. Furthermore, pull-down 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.     

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).     

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.     

The fourth transmembrane domain of the Helicobacter pylori Na+/H+ antiporter NhaA faces a water-filled channel required for ion transport

Cysteine-scanning mutagenesis was performed from Ser-130 to Leu-160 in the fourth transmembrane domain (TM4) of the Na+/H+ antiporter NhaA from Helicobacter pylori to determine the topology of each residue and to identify functionally important residues. All of the mutants were based on cysteine-less NhaA (Cys-less NhaA), which functions very similarly to the wild-type protein, and were expressed at a level similar to Cys-less NhaA. Discontinuity of [14C]N-ethylmaleimide (NEM)-reactive residues suggested that TM4 comprises residues Gly-135 to Val-156. Even within TM4, NEM reactivity was high for I136C, D141C to A143C, L146C, M150C, and G153C to R155C. These residues are thought to be located on one side of the -helical structure of TM4 and to face a putative water-filled channel. Pretreatment of intact cells with membrane-impermeable maleimide did not inhibit [14C]NEM binding to the NEM-reactive residues within TM4, suggesting that the putative channel opens toward the cytoplasm. NEM reactivity of the A143C mutant was significantly inhibited by Li+. The T140C and D141C mutants showed lower affinity for Na+ and Li+ as transport substrates, but their maximal antiporter velocities (Vmax) were relatively unaffected. Whereas the I142C and F144C mutants completely lost their Li+/H+ antiporter activity, I142C had a lower Vmax for the Na+/H+ antiporter. F144C exhibited a markedly lower Vmax and a partially reduced affinity for Na+. These results suggest that Thr-140, Asp-141, and Phe-144 are located in the end portion of a putative water-filled channel and may provide the binding site for Na+, Li+, and/or H+. Furthermore, residues Ile-142 to Phe-144 may be important for the conformational change that accompanies ion transport in NhaA.     

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.     

pH-induced structural change in a sodium/proton antiporter from Methanococcus jannaschii

Na+/H+ antiporters are pH-dependent membrane transport proteins that maintain the homeostasis of H+ and Na+ in living cells. MjNhaP1 from Methanococcus jannaschii, a hyperthermophilic archaeon that grows optimally at 85 degrees C, was cloned and expressed in Escherichia coli. Two-dimensional crystals were obtained from purified protein at pH 4. Electron cryomicroscopy yielded an 8 A projection map. Like the related E. coli antiporter NhaA, MjNhaP1 is a dimer, but otherwise the structures of the two antiporters differ significantly. The map of MjNhaP1 shows elongated densities in the centre of the dimer and a cluster of density peaks on either side of the dimer core, indicative of a bundle of 4-6 membrane-spanning helices. The effect of pH on the structure of MjNhaP1 was studied in situ. A major change in density distribution within the helix bundle, and an approximately 2 A shift in the position of the helix bundle relative to the dimer core occurred at pH 6 and above. The two conformations at low and high pH most likely represent the closed and open states of the antiporter.     

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.