NhaK,a novel monovalent cation/H<Superscript>+</Superscript> antiporter of <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Four Na⁺/H⁺ antiporters, Mrp, TetA(L), NhaC, and MleN have so far been described in Bacillus subtilis 168. We identified an additional Na⁺/H⁺ antiporter, YvgP, from B. subtilis that exhibits homology to the cation: proton antiporter-1 (CPA-1) family. The yvgP-dependent complementation observed in a Na⁺(Ca²⁺)/H⁺ antiporter-defective Escherichia coli mutant (KNabc) suggested that YvgP effluxed Na⁺ and Li⁺. In addition, effects of yvgP expression on a K⁺ uptake-defective mutant of E. coli indicated that YvgP also supported K⁺ efflux. In a fluorescence-based assay of everted membrane vesicles prepared from E. coli KNabc transformants, YvgP-dependent Na⁺ (K⁺, Li⁺, Rb⁺)/H⁺ antiport activity was demonstrated. Na⁺ (K⁺, Li⁺)/H⁺ activity was higher at pH 8.5 than at pH 7.5. Mg²⁺, Ca²⁺ and Mn²⁺ did not serve as substrates but they inhibited YvgP antiport activities. Studies of yvgP expression in B. subtilis, using a reporter gene fusion, showed a significant constitutive level of expression that was highest in stationary phase, increasing as stationary phase progressed. In addition, the expression level was significantly increased in the presence of added K⁺ and Na⁺.     

Characterization of the ion transport activity of the budding yeast Na/H antiporter, Nha1p, using isolated secretory vesicles

The Saccharomyces cerevisiae Nha1p, a plasma membrane protein belonging to the monovalent cation/proton antiporter family, plays a key role in the salt tolerance and pH regulation of cells. We examined the molecular function of Nha1p by using secretory vesicles isolated from a temperature sensitive secretory mutant, sec4-2, in vitro. The isolated secretory vesicles contained newly synthesized Nha1p en route to the plasma membrane and showed antiporter activity exchanging H⁺ for monovalent alkali metal cations. An amino acid substitution in Nha1p (D266N, Asp-266 to Asn) almost completely abolished the Na⁺/H⁺ but not K⁺/H⁺ antiport activity, confirming the validity of this assay system as well as the functional importance of Asp-266, especially for selectivity of substrate cations. Nha1p catalyzes transport of Na⁺ and K⁺ with similar affinity (12.7 mM and 12.4 mM), and with lower affinity for Rb⁺ and Li⁺. Nha1p activity is associated with a net charge movement across the membrane, transporting more protons per single sodium ion (i.e., electrogenic). This feature is similar to the bacterial Na⁺/H⁺ antiporters, whereas other known eukaryotic Na⁺/H⁺ antiporters are electroneutral. The ion selectivity and the stoichiometry suggest a unique physiological role of Nha1p which is distinct from that of other known Na⁺/H⁺ antiporters.     

K+/H+ antiport in mitochondria

Mitochondria contain a latent K⁺/H⁺ antiporter that is activated by Mg²⁺-depletion and shows optimal activity in alkaline, hypotonic suspending media. This K⁺/H⁺ antiport activity appears responsible for a respiration-dependent extrusion of endogenous K⁺, for passive swelling in K⁺ acetate and other media, for a passive exchange of matrix⁴²K⁺ against external K⁺, Na⁺, or Li⁺, and for the respiration-dependent ion extrusion and osmotic contraction of mitochondria swollen passively in K⁺ nitrate. K⁺/H⁺ antiport is inhibited by quinine and by dicyclohexylcarbodiimide when this reagent is reacted with Mg²⁺-depleted mitochondria. There is good suggestive evidence that the K⁺/H⁺ antiport may serve as the endogenous K⁺-extruding device of the mitochondrion. There is also considerable experimental support for the concept that the K⁺/H⁺ antiport is regulated to prevent futile influx-efflux cycling of K⁺. However, it is not yet clear whether such regulation depends on matrix free Mg²⁺, on membrane conformational changes, or other as yet unknown factors.     

Functional involvement of membrane-embedded and conserved acidic residues in the ShaA subunit of the multigene-encoded Na/H antiporter in Bacillus subtilis

ShaA, a member of a multigene-encoded Na⁺/H⁺ antiporter in B. subtilis, is a large integral membrane protein consisting of 20 transmembrane helices (TM). Conservation of ShaA-like protein subunits in several cation-coupled enzymes, including the NuoL (ND5) subunit of the H⁺-translocating complex I, suggests the involvement of ShaA in cation transport. Bacillus subtilis ShaA contains six acidic residues that are conserved in ShaA homologues and are located in putative transmembrane helices. We examined the functional involvement of the six transmembrane acidic residues of ShaA by site-directed mutagenesis. Mutation in glutamate (Glu)-113 in TM-4, Glu-657 in TM-18, aspartate (Asp)-734 and Glu-747 in TM-20 abolished the antiport activity, suggesting that these residues play important roles in the ion transport of Sha. The acidic group was necessary and sufficient in Glu-657 and Asp-743, while it was not true of Glu-113 and Glu-747. Mutation in Asp-103 in TM-3, which is conserved in ShaA-types but not in ShaAB-types, partially affected on the antiport activity. Mutation in Asp-50 in TM-2 resulted in a unexpected phenotype: mutants retained the wild type level of ability to confer NaCl resistance to the Na⁺/H⁺ antiporter-deficient E. coli KNabc, but showed a very low antiport activity. The acidic group of Asp-50 and Asp-103 was not essential for the function. Our results suggested that these acidic residues are functionally involved in the ion transport of Sha, and some of them probably in cation binding and/or translocation.     

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.     

Cloning,sequencing and expression of the nhaA and nhaR genes from Salmonella entiritidis

Na⁺/H⁺ antiporter activity is wide-spread and plays essential physiological roles. We found that several Enterobacteriaceae share conserved sequences with nhaA, the gene coding for an E. coli antiporter. A nhaA strain which is sensitive to Na⁺ and Li⁺, was used to clone by complementation a DNA fragment from Salmonella enteritidis which confers resistance to the ions. The cloned fragment increased Na⁺/H⁺ antiport activity in membranes isolated from strains carrying the respective hybrid plasmid. DNA sequence analysis of the insert revealed two open reading frames. Both encode putative polypeptides which are closely homologous to the nhaA and nhaR gene products from Escherichia coli. The antiporter activity displays properties very similar to that of the E. coli NhaA, namely, it is activiated by alkaline pH and recognizes Li⁺ with high affinity.Abbreviations _H ⁺ Proton electrochemical potential - pH transmembrane pH gradient - _Na ⁺ Sodium electrochemical potential - SDS Sodium dodecyl sulfate - CIP Calf intestine alkaline phosphates - ORF open reading frame     

Ion Specificity of Na+-Transporting Systems in the Plasma Membrane of the Halotolerant Alga Tetraselmis (Platymonas) viridis

Vesicular preparations of plasma membranes (PM) from the microalga Tetraselmis (Platymonas) viridisRouch were used to investigate the ion specificity of the Na⁺/H⁺antiporter and Na⁺-translocating ATPase, two Na⁺-transporting systems previously identified functionally by our studies of T. viridisPM. The Na⁺/H⁺antiporter and Na⁺-ATPase were shown to translocate, with similar efficiencies, Na⁺and Li⁺across the membrane, whereas other cations, such as K⁺, Rb⁺, and Cs⁺, were not transported by these systems. Transport of the latter cations across PM of T. viridisoccurred through the ion channels of PM, which were apparently selective for K⁺.     

Respiration-dependent contraction of swollen heart mitochondria: Participation of the K+/H+ antiporter

Respiration-dependent contraction of heart mitochondria swollen passively in K⁺ nitrate is activated by the ionophore A23187 and inhibited by Mg²⁺. Ion extrusion and osmotic contraction under these conditions are strongly inhibited by quinine, a known inhibitor of the mitochondrial K⁺/H⁺ antiporter, as measured in other systems. The inhibition by quinine is relieved by the exogenous antiporter nigericin. Respiration-dependent contraction is also inhibited by dicyclohexylcarbodiimide (DCCD) when reacted under conditions known to inhibit K⁺/H⁺ antiport (Martinet al., J. Biol. Chem. 259, 2062–2065, 1984). These studies strongly support the concept that K⁺ is extruded from the matrix by the endogenous K⁺/H⁺ antiporter and that inhibition of this component by quinine or DCCD inhibits respiration-dependent contraction. The extrusion of K⁺ nitrate is accompanied by a respiration-dependent efflux of a considerable portion of the endogenous Mg²⁺. This Mg²⁺ efflux does not occur in the presence of nigericin or when the mitochondrial Na⁺/H⁺ antiporter is active. Mg²⁺ efflux may take place on the K⁺/H⁺ antiporter. DCCD, reacted under conditions that do not result in inhibition of the K⁺/H⁺ antiporter, blocks a monovalent cation uniport pathway. This uniport contributes to futile cation cycling at elevated pH, and its inhibition by DCCD stimulates respiration-dependent contraction.     

Functional Identification of H<Superscript>+</Superscript>-ATPase and Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter in the Plasma Membrane Isolated from the Root Cells of Salt-Accumulating Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis>

A membrane fraction enriched in plasma membrane (PM) vesicles was isolated from the root cells of a salt-accumulating halophyte Suaeda altissima (L.) Pall. by means of centrifugation in discontinuous sucrose density gradient. The PM vesicles were capable of generating ΔpH at their membrane and the transmembrane electric potential difference (Δψ). These quantities were measured with optical probes, acridine orange and oxonol VI, sensitive to ΔpH and Δψ, respectively. The ATP-dependent generation of ΔpH was sensitive to vanadate, an inhibitor of P-type ATPases. The results contain evidence for the functioning of H⁺-ATPase in the PM of the root cells of S. altissima. The addition of Na⁺ and Li⁺ ions to the outer medium resulted in dissipation of ΔpH preformed by the H⁺-ATPase, which indicates the presence in PM of the functionally active Na⁺/H⁺ antiporter. The results are discussed with regard to involvement of the Na⁺/H⁺ antiporter and the PM H⁺-ATPase in loading Na⁺ ions into the xylem of S. altissima roots.     

A nhaD Na/Hantiporter and a pcd homologues are among the Rhodothermus marinus complex I genes

The NADH:menaquinone oxidoreductase (Nqo) is one of the enzymes present in the respiratory chain of the thermohalophilic bacterium Rhodothermus marinus. The genes coding for the R. marinus Nqo subunits were isolated and sequenced, clustering in two operons [nqo₁ to nqo₇ (nqo_A) and nqo₁₀ to nqo₁₄ (nqo_B)] and two independent genes (nqo₈ and nqo₉). Unexpectedly, two genes encoding homologues of a NhaD Na⁺/H⁺ antiporter (NhaD) and of a pterin-4α-carbinolamine dehydratase (PCD) were identified within nqo_B, flanked by nqo₁₃ and nqo₁₄. Eight conserved motives to harbour iron-sulphur centres are identified in the deduced primary structures, as well as two consensus sequences to bind nucleotides, in this case NADH and FMN. Moreover, the open-reading-frames of the putative NhaD and PCD were shown to be co-transcribed with the other complex I genes encoded by nqo_B. The possible role of these two genes in R. marinus complex I is discussed.     

Role of the Na+/H+ antiport in the regulation of the internal pH of Ehrlich ascites tumor cells in culture

Summary Ehrlich ascites tumor cells contain a Na⁺ uptake system, which is activated by internal protons and is inhibited by amiloride with an IC₅₀ of 25 m and by dimethylamiloride with an IC₅₀ of 0.6 m at 1mm external Na⁺. Decrease of external Na⁺ or addition of amiloride is followed by a decrease of internal pH. Taken together, these findings suggest the presence of an operative Na⁺/H⁺ antiport system, which is involved in the regulation of internal pH. We cannot find a significant contribution of a proton pump activated by glycolysis to the pH gradient. At an external pH between 7.0 and 7.6, quiescent cells are more alkaline than exponentially growing cells (0.1 to 0.17 units). Accordingly, an increase of the affinity of the Na⁺/H⁺ antiport for internal protons in quiescent cells is demonstrated by the following findings: 1. The internal pH, at which the half-maximal activation of the amiloride-sensitive Na⁺ uptake occurs, is shifted from 6.85 to 7.1 at 1mm external Na⁺. 2. The threshold value of external pH, below which a pronounced effect of amiloride on steadystate internal pH is observed, is shifted from 7.0 in growing to 7.5 in quiescent cells at physiological Na⁺ concentrations. Therefore, we conclude that quiescent Ehrlich ascites tumor cells raise their internal pH by increasing the affinity of their Na⁺/H⁺ antiporter to internal protons. The Na⁺/H⁺ antiport cannot be activated further by addition of serum growth factors to quiescent cells. All experiments were performed at bicarbonate concentrations in the medium which do not exceed 0.5mm. The data are discussed in view of existing models of mitogenic activity of transitory pH changes.     

Molecular Cloning and Functional Analysis of a Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter Gene <Emphasis Type="Italic">ThNHX1</Emphasis> from a Halophytic Plant <Emphasis Type="Italic">Thellungiella halophila</Emphasis>

Na⁺/H⁺ exchanger catalyzes the countertransport of Na⁺ and H⁺ across membranes. Using the rapid amplification of cDNA ends method, a Na⁺/H⁺ antiporter gene (ThNHX1) was isolated from a halophytic plant, salt cress (Thellungiella halophila). The deduced amino acid sequence contained 545 amino acid residues with a conserved amiloride-binding domain (⁸⁷LFFIYLLPPI⁹⁶) and shared more than 94% identity with that of AtNHX1 from Arabidopsis thaliana. The ThNHX1 mRNA level was upregulated by salt and other stresses (abscisic acid, polyethylene glycol, and high temperature). This gene partially complemented the Na⁺/Li⁺-sensitive phenotype of a yeast mutant that was deficient in the endosomal–vacuolar Na⁺/H⁺ antiporter ScNHX1. Overexpression of ThNHX1 in Arabidopsis increased salt tolerance of transgenic plants compared with the wild-type plants. In addition, the silencing of ThNHX1 gene in T. halophila caused the transgenic plants to be more salt and osmotic sensitive than wild-type plant. Together, these results suggest that ThNHX1 may function as a tonoplast Na⁺/H⁺ antiporter and play an important role in salt tolerance of T. halophila. Chunxia Wu, Xiuhua Gao, and Xiangqiang Kong contributed equally to this work.     

ATP Dependence of Na+/H+ Exchange : Nucleotide Specificity and Assessment of the Role of Phospholipids

We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na⁺/H⁺ antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pH_i) by microfluorimetry. Na⁺/H⁺ exchange activity was measured as the Na⁺-driven pH_i recovery from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably transfected with NHE-1, omission of ATP from the pipette solution inhibited Na⁺/H⁺ exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-d-glucose and oligomycin. In cells dialyzed in the presence of ATP, no “run-down” was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of the antiporter was obtained at ∼5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na⁺/H⁺ exchange even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca²⁺ exchanger, this requirement has been attributed to an aminophospholipid translocase, or “flippase.” The involvement of this enzyme in Na⁺/H⁺ exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na⁺/H⁺ exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATPγS were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted for ATP. In contrast, GTPγS was ineffective. We conclude that ATP is the only soluble factor necessary for optimal activity of the NHE-1 isoform of the antiporter. Mg²⁺ does not appear to be essential for the stimulatory effect of ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis and is likely an energy-dependent event. The second process does not involve hydrolysis of the γ-phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter.     

A Novel Plant Vacuolar Na+/H+ Antiporter Gene Evolved by DNA Shuffling Confers Improved Salt Tolerance in Yeast

Plant vacuolar Na⁺/H⁺ antiporters play important roles in maintaining cellular ion homeostasis and mediating the transport of Na⁺ out of the cytosol and into the vacuole. Vacuolar antiporters have been shown to play significant roles in salt tolerance; however the relatively low V_max of the Na⁺/H⁺ exchange of the Na⁺/H⁺ antiporters identified could limit its application in the molecular breeding of salt tolerant crops. In this study, we applied DNA shuffling methodology to generate and recombine the mutations of Arabidopsis thaliana vacuolar Na⁺/H⁺ antiporter gene AtNHX1. Screening using a large scale yeast complementation system identified AtNHXS1, a novel Na⁺/H⁺ antiporter. Expression of AtNHXS1 in yeast showed that the antiporter localized to the vacuolar membrane and that its expression improved the tolerance of yeast to NaCl, KCl, LiCl, and hygromycin B. Measurements of the ion transport activity across the intact yeast vacuole demonstrated that the AtNHXS1 protein showed higher Na⁺/H⁺ exchange activity and a slightly improved K⁺/H⁺ exchange activity.     

Protective effect of exogenous polyamines on root tonoplast function against salt stress in barley seedlings

Salinity stress is one of the most serious factors limiting the productivity of agricultural crops. A possible survival strategy of plants under saline conditions is to sequester excess Na⁺ in the vacuole by vacuolar Na⁺/H⁺ antiport using a pH gradient generated by H⁺-ATPasc (EC 3.6.1.35) and H⁺-Pyrophosphatase (H⁺-PPase; EC 3.6.1.1) to maintain a higher K⁺/Na⁺ ratio in cytoplasm. The effect of exogenously applied polyamines (PAs) in stabilizing root tonoplast integrity and function against salt stress in the barley (Hordeum vulgare L.) seedlings was investigated. The NaCl-induced reductions in the contents of phospholipids and PAs in tonoplast vesicles isolated from barely seedling roots, as well as the activities of H⁺-ATPase, H⁺-PPase and vacuolar Na⁺/H⁺ antiport were all partially restored by the application of 0.5 mM putrescine and 0.5 mM spermidine, especially the former. The above results indicated that one of the mechanisms involved in attenuating salt injury in barley seedlings by exogenous PAs application was to maintain tonoplast integrity and function under saline conditions. Moreover, the possible mechanism involved in counteracting detrimental effects of salt on the barley seedlings by the application of exogenous PAs was discussed.     

Sodium ATPase and Sodium/proton Antiporter Are Not Obligatory for Sodium Homeostasis of Enterococcus hirae at Acid pH

Enterococcus hirae grows in a broad pH range from 5 to 11. An E. hirae mutant 7683 lacking the activities of two sodium pumps, Na⁺-ATPase and Na⁺/H⁺ antiporter, does not grow in high Na⁺ medium at pH above 7.5. We found that 7683 grew normally in high Na⁺ medium at pH 5.5. Although an energy-dependent sodium extrusion at pH 5.5 was missing, the intracellular levels of Na⁺ and K⁺ were normal in this mutant. The Na⁺ influx rates of 7683 and two other strains at pH 5.5 were much slower than those at pH 7.5. These results suggest that Na⁺ elimination of this bacterium at acid pH is achieved by a decrease in Na⁺ entry and a normal K⁺ uptake.     

Mutational analysis of potential pore-lining amino acids in TM IV of the Na/H exchanger

The Na⁺/H⁺ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. In this study we examined the effect of site-specific mutagenesis on the pore-lining amino acid Phe161 and effects of mutagenesis on the charged amino acids Asp159 and Asp172. There was no absolute requirement for a carboxyl side chain at amino acid Asp159 or Asp172. Mutation of Asp159 to Asn or Gln maintained or increased the activity of the protein. Similarly, for Asp172, substitution with a Gln residue maintained activity of the protein, even though substitution with an Asn residue was inhibitory. The Asp172Glu mutant possessed normal activity after correction for its aberrant expression and surface targeting. Replacement of Phe161 with a Leu demonstrated that it was not irreplaceable in NHE1 function. However, the mutation Phe161lys inhibited NHE1 function, while the Phe161Ala mutation caused altered NHE1 targeting and expression levels. Our results show that these three amino acids, while being important in NHE1 function, are not irreplaceable. This study demonstrates that multiple substitutions at a single amino acid residue may be necessary to get a clearer picture membrane protein function.     

Conformational changes in NhaA Na+/H+ antiporter

Abstract

Na⁺/H⁺ antiporters play a primary role in Na⁺/H⁺ homeostasis in cells and many organelles and have long been drug targets. The X-ray structure of NhaA, the main antiporter of Escherichia coli, provided structural insights into the antiport mechanism and its pH regulation and revealed a novel fold; six of the 12 TMs (Trans membrane segments) are organized in two topologically inverted repeats, each with one TM interrupted by an extended chain creating a unique electrostatic environment in the middle of the membrane at the cation binding site. Remarkably, inverted repeats containing interrupted helices with similar functional implications have since been observed in structures of other bacterial secondary transporters with almost no sequence homology. Finally, the structure reveals that NhaA is organized into two functional regions: a ‘pH sensor' – a cluster of amino acyl side chains that are involved in pH regulation; and a catalytic region that is 9 Å removed from the pH sensor. Alternative accessibility of the binding site to either side of the membrane, i.e., functional-dynamics, is the essence of secondary transport mechanism. Because NhaA is tightly pH regulated, structures of the pH-activated and ligand-activated NhaA conformations are needed to identify its functional-dynamics. However, as these are static snapshots of a dynamic protein, the dynamics of the protein both in vitro and in situ in the membrane are also required as reviewed here in detail. The results reveal two different conformational changes characterizing NhaA: One is pH-induced for NhaA activation; the other is ligand-induced for antiport activity.