Unconventional Chemiosmotic Coupling of NHA2, a Mammalian Na+/H+ Antiporter,to a Plasma Membrane H+ Gradient

Human NHA2, a newly discovered cation proton antiporter, is implicated in essential hypertension by gene linkage analysis. We show that NHA2 mediates phloretin-sensitive Na⁺-Li⁺ counter-transport (SLC) activity, an established marker for hypertension. In contrast to bacteria and fungi where H⁺ gradients drive uptake of metabolites, secondary transport at the plasma membrane of mammalian cells is coupled to the Na⁺ electrochemical gradient. Our findings challenge this paradigm by showing coupling of NHA2 and V-type H⁺-ATPase at the plasma membrane of kidney-derived MDCK cells, resulting in a virtual Na⁺ efflux pump. Thus, NHA2 functionally recapitulates an ancient shared evolutionary origin with bacterial NhaA. Although plasma membrane H⁺ gradients have been observed in some specialized mammalian cells, the ubiquitous tissue distribution of NHA2 suggests that H⁺-coupled transport is more widespread. The coexistence of Na⁺ and H⁺-driven chemiosmotic circuits has implications for salt and pH regulation in the kidney.     

Computational study of the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter from <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

Sodium proton antiporters are ubiquitous membrane proteins that catalyze the exchange of Na⁺ for protons throughout the biological world. The Escherichia coli NhaA is the archetypal Na⁺/H⁺ antiporter and is absolutely essential for survival in high salt concentrations under alkaline conditions. Its crystal structure, accompanied by extensive molecular dynamics simulations, have provided an atomically detailed model of its mechanism. In this study, we utilized a combination of computational methodologies in order to construct a structural model for the Na⁺/H⁺ antiporter from the gram-negative bacterium Vibrio parahaemolyticus. We explored its overall architecture by computational means and validated its stability and robustness. This protein belongs to a novel group of NhaA proteins that transports not only Na⁺ and Li⁺ as substrate ions, but K⁺ as well, and was also found to miss a β-hairpin segment prevalent in other homologs of the Bacteria domain. We propose, for the first time, a structure of a prototype model of a β-hairpin-less NhaA that is selective to K⁺. Better understanding of the Vibrio parahaemolyticus NhaA structure-function may assist in studies on ion transport, pH regulation and designing selective blockers.     

Functional Analysis of the Na+,K+/H+ Antiporter PeNHX3 from the Tree Halophyte Populus euphratica in Yeast by Model-Guided Mutagenesis

Na⁺,K⁺/H⁺ antiporters are H⁺-coupled cotransporters that are crucial for cellular homeostasis. Populus euphratica, a well-known tree halophyte, contains six Na⁺/H⁺ antiporter genes (PeNHX1-6) that have been shown to function in salt tolerance. However, the catalytic mechanisms governing their ion transport remain largely unknown. Using the crystal structure of the Na⁺/H⁺ antiporter from the Escherichia coli (EcNhaA) as a template, we built the three-dimensional structure of PeNHX3 from P. euphratica. The PeNHX3 model displays the typical TM4-TM11 assembly that is critical for ion binding and translocation. The PeNHX3 structure follows the ‘positive-inside’ rule and exhibits a typical physicochemical property of the transporter proteins. Four conserved residues, including Tyr149, Asn187, Asp188, and Arg356, are indentified in the TM4-TM11 assembly region of PeNHX3. Mutagenesis analysis showed that these reserved residues were essential for the function of PeNHX3: Asn187 and Asp188 (forming a ND motif) controlled ion binding and translocation, and Tyr149 and Arg356 compensated helix dipoles in the TM4-TM11 assembly. PeNHX3 mediated Na⁺, K⁺ and Li⁺ transport in a yeast growth assay. Domain-switch analysis shows that TM11 is crucial to Li⁺ transport. The novel features of PeNHX3 in ion binding and translocation are discussed.     

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.     

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 K_m 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 K_m as well as turnover (V_max).     

Mutational analysis of NHAoc/NHA2 in Saccharomyces cerevisiae

Background

NHAoc/NHA2 is highly and selectively expressed in osteoclasts and plays a role(s) in normal osteoclast differentiation, apoptosis and bone resorptive function in vitro. Extensive mutational analysis of a bacterial homologue, NhaA, has revealed a number of amino acid residues essential for its activity. Some of these residues are evolutionarily conserved and have been shown to be essential not only for activity of NhaA in bacteria, but also of NHAoc/NHA2 in eukaryotes.

Methods

The salt-sensitive Saccharomyces cerevisiae strain BW31a was used for heterologous expression of mutants of NHAoc/NHA2. Membrane expression of NHAoc/NHA2 was confirmed by confocal microscopy. Intracellular concentration of Na+ (a measure of Na+ antiporter activity) was estimated by atomic absorption spectroscopy. The growth phenotypes of cells expressing NHAoc/NHA2 mutants were studied on YNB agar supplemented with NaCl and by growth curves in YNB broth.

Results

Mutations in amino acid residues V161 and F357 reduced the ability of transfected BW31a cells to remove intracellular sodium and to grow in NaCl-containing medium. Yeast expressing the double mutant F357 F437 cannot grow in 0.4 M NaCl, suggesting that these residues are also essential for antiporter activity.

Conclusions

Evolutionarily conserved amino acids are required for full antiporter function.

General Significance

Mutations in these amino acid residues may impact NHAoc activity and therefore osteoclast function in vitro and in vivo.     

Helix VIII of NhaA Na(+)/H(+) antiporter participates in the periplasmic cation passage and pH regulation 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 active at physiological pH (pH 6.5-8.5), many questions related to the active state of NhaA have remained unanswered. Our Cys scanning of the highly conserved transmembrane VIII at physiological pH reveals that (1) the Cys replacement G230C significantly increases the apparent K_m of the antiporter to both Na⁺ (10-fold) and Li⁺ (6-fold). (2) Variants G223C and G230C cause a drastic alkaline shift of the pH profile of NhaA by 1 pH unit. (3) Residues Gly223-Ala226 line a periplasmic funnel at physiological pH as they do at pH 4. Both were modified by membrane-impermeant negatively charged 2-sulfonatoethyl methanethiosulfonate and positively charged 2-(trimethyl ammonium)-ethylmethanethiosulfonate sulfhydryl reagents that could reach Cys replacements from the periplasm via water-filled funnels only, whereas other Cys replacements on helix VIII were not accessible/reactive to the reagents. (4) Remarkably, the modification of variant V224C by 2-sulfonatoethyl methanethiosulfonate or 2-(trimethyl ammonium)-ethylmethanethiosulfonate totally inhibited antiporter activity, while N-ethyl maleimide modification had a very small effect on NhaA activity. Hence, the size—rather than the chemical modification or the charge—of the larger reagents interferes with the passage of ions through the periplasmic funnel. Taken together, our results at physiological pH reveal that amino acid residues in transmembrane VIII contribute to the cation passage of NhaA and its pH regulation.     

Functional and structural dynamics of NhaA,a prototype for Na and H antiporters,which are responsible for Na and H homeostasis in cells

The crystal structure of down-regulated NhaA crystallized at acidic pH 4 [21] has provided the first structural insights into the antiport mechanism and pH regulation of a Na⁺/H⁺ antiporter [22]. On the basis of the NhaA crystal structure [21] and experimental data (reviewed in [2,22,38] we have suggested that NhaA is organized into two functional regions: (i) a cluster of amino acids responsible for pH regulation (ii) a catalytic region at the middle of the TM IV/XI assembly, with its unique antiparallel unfolded regions that cross each other forming a delicate electrostatic balance in the middle of the membrane. This unique structure contributes to the cation binding site and allows the rapid conformational changes expected for NhaA. Extended chains interrupting helices appear now a common feature for ion binding in transporters. However the NhaA fold is unique and shared by ASBTNM [30] and NapA [29]. Computation [13], electrophysiology [69] combined with biochemistry [33,47] have provided intriguing models for the mechanism of NhaA. However, the conformational changes and the residues involved have not yet been fully identified. Another issue which is still enigma is how energy is transduced “in this ‘nano-machine.’” We expect that an integrative approach will reveal the residues that are crucial for NhaA activity and regulation, as well as elucidate the pHand ligand-induced conformational changes and their dynamics. Ultimately, integrative results will shed light on the mechanism of activity and pH regulation of NhaA, a prototype of the CPA2 family of transporters. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

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.     

Differential Effects of Mutations on the Transport Properties of the Na+/H+ Antiporter NhaA from Escherichia coli

Na⁺/H⁺ antiporters show a marked pH dependence, which is important for their physiological function in eukaryotic and prokaryotic cells. In NhaA, the Escherichia coli Na⁺/H⁺ antiporter, specific single site mutations modulating the pH profile of the transporter have been described in the past. To clarify the mechanism by which these mutations influence the pH dependence of NhaA, the substrate dependence of the kinetics of selected NhaA variants was electrophysiologically investigated and analyzed with a kinetic model. It is shown that the mutations affect NhaA activity in quite different ways by changing the properties of the binding site or the dynamics of the transporter. In the first case, pK and/or K_D^Na are altered, and in the second case, the rate constants of the conformational transition between the inside and the outside open conformation are modified. It is shown that residues as far apart as 15–20 Å from the binding site can have a significant impact on the dynamics of the conformational transitions or on the binding properties of NhaA. The implications of these results for the pH regulation mechanism of NhaA are discussed.     

NhaA Na+/H+ Antiporter Mutants That Hardly React to the Membrane Potential

pH and Na⁺ homeostasis in all cells requires Na⁺/H⁺ antiporters. The crystal structure, obtained at pH 4, of NhaA, the main antiporter of Escherichia coli, has provided general insights into an antiporter mechanism and its unique pH regulation. Here, we describe a general method to select various NhaA mutants from a library of randomly mutagenized NhaA. The selected mutants, A167P and F267C are described in detail. Both mutants are expressed in Escherichia coli EP432 cells at 70–95% of the wild type but grow on selective medium only at neutral pH, A167P on Li⁺ (0.1 M) and F267C on Na⁺ (0.6 M). Surprising for an electrogenic secondary transporter, and opposed to wild type NhaA, the rates of A167P and F267C are almost indifferent to membrane potential. Detailed kinetic analysis reveals that in both mutants the rate limiting step of the cation exchange cycle is changed from an electrogenic to an electroneutral reaction.     

Towards Molecular Understanding of the pH Dependence Characterizing NhaA of Which Structural Fold is Shared by Other Transporters

Na⁺/H⁺ antiporters comprise a super-family (CPA) of membrane proteins that are found in all kingdoms of life and are essential in cellular homeostasis of pH, Na⁺ and volume. Their activity is strictly dependent on pH, a property that underpins their role in pH homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystal structure provided insight into the architecture of this molecular machine. However, the mechanism of the strict pH dependence of NhaA is missing. Here, as a follow up of a recent evolutionary analysis that identified a ‘CPA motif’, we rationally designed three E. coli NhaA mutants: D133S, I134T, and the double mutant D133S-I134T. Exploring growth phenotype, transport activity and Li⁺-binding of the mutants, we revealed that Asp133 does not participate directly in proton binding, nor does it directly dictate the pH-dependent transport of NhaA. Strikingly, the variant I134T lost some of the pH control, and the D133S-Il134T double mutant retained Li⁺ binding in a pH independent fashion. Concurrent to loss of pH control, these mutants bound Li⁺ more strongly than the WT. Both positions are in close vicinity to the ion-binding site of the antiporter, attributing the results to electrostatic interaction between these residues and Asp164 of the ion-binding site. This is consistent with pH sensing resulting from direct coupling between cation binding and deprotonation in Asp164, which applies also to other CPA antiporters that are involved in human diseases.     

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.     

Crystal structure of the sodium–proton antiporter NhaA dimer and new mechanistic insights

Sodium–proton antiporters rapidly exchange protons and sodium ions across the membrane to regulate intracellular pH, cell volume, and sodium concentration. How ion binding and release is coupled to the conformational changes associated with transport is not clear. Here, we report a crystal form of the prototypical sodium–proton antiporter NhaA from Escherichia coli in which the protein is seen as a dimer. In this new structure, we observe a salt bridge between an essential aspartic acid (Asp163) and a conserved lysine (Lys300). An equivalent salt bridge is present in the homologous transporter NapA, but not in the only other known crystal structure of NhaA, which provides the foundation of most existing structural models of electrogenic sodium–proton antiport. Molecular dynamics simulations show that the stability of the salt bridge is weakened by sodium ions binding to Asp164 and the neighboring Asp163. This suggests that the transport mechanism involves Asp163 switching between forming a salt bridge with Lys300 and interacting with the sodium ion. pK_a calculations suggest that Asp163 is highly unlikely to be protonated when involved in the salt bridge. As it has been previously suggested that Asp163 is one of the two residues through which proton transport occurs, these results have clear implications to the current mechanistic models of sodium–proton antiport in NhaA.     

Revealing the Ligand Binding Site of NhaA Na+/H+ Antiporter and Its pH Dependence

pH and Na⁺ homeostasis in all cells requires Na⁺/H⁺ antiporters. In most cases, their activity is tightly pH-regulated. NhaA, the main antiporter of Escherichia coli, has homologues in all biological kingdoms. The crystal structure of NhaA provided insights into the mechanism of action and pH regulation of an antiporter. However, the active site of NhaA remained elusive because neither Na⁺ nor Li⁺, the NhaA ligands, were observed in the structure. Using isothermal titration calorimetry, we show that purified NhaA binds Li⁺ in detergent micelles. This interaction is driven by an increase in enthalpy (ΔH of −8000 ± 300 cal/mol and ΔS of −15.2 cal/mol/degree at 283 K), involves a single binding site per NhaA molecule, and is highly specific and drastically dependent on pH; Li⁺ binding was observed only at pH 8.5. Combining mutational analysis with the isothermal titration calorimetry measurements revealed that Asp-163, Asp-164, Thr-132, and Asp-133 form the Li⁺ binding site, whereas Lys-300 plays an important role in pH regulation of the antiporter.     

Asp133 Residue in NhaA Na+/H+ Antiporter Is Required for Stability Cation Binding and Transport

Na⁺/H⁺ antiporters have a crucial role in pH and Na⁺ homeostasis in cells. The crystal structure of NhaA, the main antiporter of Escherichia coli, has provided general insights into antiporter mechanisms and revealed a previously unknown structural fold, which has since been identified in several secondary active transporters. This unique structural fold is very delicately electrostatically balanced. Asp133 and Lys 300 have been ascribed essential roles in this balance and, more generally, in the structure and function of the antiporter. In this work, we show the multiple roles of Asp133 in NhaA: (i) The residue's negative charge is critical for the stability of the NhaA structure. (ii) Its main chain is part of the active site. (iii) Its side chain functions as an alkaline-pH-dependent gate, changing the protein's conformation from an inward-facing conformation at acidic pH to an outward-open conformation at alkaline pH, opening the periplasm funnel. On the basis of the experimental data, we propose a tentative mechanism integrating the structural and functional roles of Asp133.     

Molecular Characterization of the Na+/H+-Antiporter NhaA from Salmonella Typhimurium

Na⁺/H⁺ antiporters are integral membrane proteins that are present in almost every cell and in every kingdom of life. They are essential for the regulation of intracellular pH-value, Na⁺-concentration and cell volume. These secondary active transporters exchange sodium ions against protons via an alternating access mechanism, which is not understood in full detail. Na⁺/H⁺ antiporters show distinct species-specific transport characteristics and regulatory properties that correlate with respective physiological functions. Here we present the characterization of the Na⁺/H⁺ antiporter NhaA from Salmonella enterica serovar Thyphimurium LT2, the causing agent of food-born human gastroenteritis and typhoid like infections. The recombinant antiporter was functional in vivo and in vitro. Expression of its gene complemented the Na⁺-sensitive phenotype of an E. coli strain that lacks the main Na⁺/H⁺ antiporters. Purified to homogeneity, the antiporter was a dimer in solution as accurately determined by size-exclusion chromatography combined with multi-angle laser-light scattering and refractive index monitoring. The purified antiporter was fully capable of electrogenic Na⁺(Li⁺)/H⁺-antiport when reconstituted in proteoliposomes and assayed by solid-supported membrane-based electrophysiological measurements. Transport activity was inhibited by 2-aminoperimidine. The recorded negative currents were in agreement with a 1Na⁺(Li⁺)/2H⁺ stoichiometry. Transport activity was low at pH 7 and up-regulation above this pH value was accompanied by a nearly 10-fold decrease of K_m ^Na (16 mM at pH 8.5) supporting a competitive substrate binding mechanism. K⁺ does not affect Na⁺ affinity or transport of substrate cations, indicating that selectivity of the antiport arises from the substrate binding step. In contrast to homologous E. coli NhaA, transport activity remains high at pH values above 8.5. The antiporter from S. Typhimurium is a promising candidate for combined structural and functional studies to contribute to the elucidation of the mechanism of pH-dependent Na⁺/H⁺ antiporters and to provide insights in the molecular basis of species-specific growth and survival strategies.     

Insights into the mechanisms of transport and regulation of the arabidopsis high-affinity K+ transporter HAK51

The high-affinity K⁺ transporter HAK5 from Arabidopsis (Arabidopsis thaliana) is essential for K⁺ acquisition and plant growth at low micromolar K⁺ concentrations. Despite its functional relevance in plant nutrition, information about functional domains of HAK5 is scarce. Its activity is enhanced by phosphorylation via the AtCIPK23/AtCBL1-9 complex. Based on the recently published three-dimensionalstructure of the bacterial ortholog KimA from Bacillus subtilis, we have modeled AtHAK5 and, by a mutational approach, identified residues G67, Y70, G71, D72, D201, and E312 as essential for transporter function. According to the structural model, residues D72, D201, and E312 may bind K⁺, whereas residues G67, Y70, and G71 may shape the selective filter for K⁺, which resembles that of K⁺shaker-like channels. In addition, we show that phosphorylation of residue S35 by AtCIPK23 is required for reaching maximal transport activity. Serial deletions of the AtHAK5 C-terminus disclosed the presence of an autoinhibitory domain located between residues 571 and 633 together with an AtCIPK23-dependent activation domain downstream of position 633. Presumably, autoinhibition of AtHAK5 is counteracted by phosphorylation of S35 by AtCIPK23. Our results provide a molecular model for K⁺ transport and describe CIPK-CBL-mediated regulation of plant HAK transporters.

Structure-function analysis of a high-affinity root K⁺ transporter reveals residues involved in transport, regulation by a protein kinase, and autoinhibition.     

Examining the dynamic energy landscape of an antiporter upon inhibitor binding

Previously, we applied single-molecule force spectroscopy to detect and locate interactions within the functional Na⁺/H⁺ antiporter NhaA from Escherichia coli. It was observed that the binding of the inhibitor 2-aminoperimidine established interactions different from those introduced by the binding of the native ligand. To understand the inhibitory mechanism of the inhibitor, we applied single-molecule dynamic force spectroscopy to reconstruct the energy landscape of NhaA. Dynamic force spectroscopy revealed that the energy landscape of the antiporter remained mainly unchanged except for the energy barrier of the functionally important transmembrane α-helix IX. Inhibitor binding set this domain into a newly formed deep and narrow energy minimum that kinetically stabilized α-helix IX and reduced its conformational entropy. The entropy reduction of α-helix IX is thought to inhibit its functionally important structural flexibility, while the deeper energy barrier shifted the population of active antiporters towards inhibited antiporters.