The Membrane Subunit NuoL(ND5) Is Involved in the Indirect Proton Pumping Mechanism of Escherichia coli Complex I

Complex I pumps protons across the membrane by using downhill redox energy. Here, to investigate the proton pumping mechanism by complex I, we focused on the largest transmembrane subunit NuoL (Escherichia coli ND5 homolog). NuoL/ND5 is believed to have H⁺ translocation site(s), because of a high sequence similarity to multi-subunit Na⁺/H⁺ antiporters. We mutated thirteen highly conserved residues between NuoL/ND5 and MrpA of Na⁺/H⁺ antiporters in the chromosomal nuoL gene. The dNADH oxidase activities in mutant membranes were mostly at the control level or modestly reduced, except mutants of Glu-144, Lys-229, and Lys-399. In contrast, the peripheral dNADH-K₃Fe(CN)₆ reductase activities basically remained unchanged in all the NuoL mutants, suggesting that the peripheral arm of complex I was not affected by point mutations in NuoL. The proton pumping efficiency (the ratio of H⁺/e⁻), however, was decreased in most NuoL mutants by 30–50%, while the IC₅₀ values for asimicin (a potent complex I inhibitor) remained unchanged. This suggests that the H⁺/e⁻ stoichiometry has changed from 4H⁺/2e⁻ to 3H⁺ or 2H⁺/2e⁻ without affecting the direct coupling site. Furthermore, 50 μm of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), a specific inhibitor for Na⁺/H⁺ antiporters, caused a 38 ± 5% decrease in the initial H⁺ pump activity in the wild type, while no change was observed in D178N, D303A, and D400A mutants where the H⁺ pumping efficiency had already been significantly decreased. The electron transfer activities were basically unaffected by EIPA in both control and mutants. Taken together, our data strongly indicate that the NuoL subunit is involved in the indirect coupling mechanism.     

Transport of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript> by an antiporter-related subunit from the <Emphasis Type="Italic">Escherichia coli </Emphasis>NADH dehydrogenase I produced in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The NADH dehydrogenase I from Escherichia coli is a bacterial homolog of the mitochondrial complex I which translocates Na⁺ rather than H⁺. To elucidate the mechanism of Na⁺ transport, the C-terminally truncated NuoL subunit (NuoL_N) which is related to Na⁺/H⁺ antiporters was expressed as a protein A fusion protein (ProtA–NuoL_N) in the yeast Saccharomyces cerevisiae which lacks an endogenous complex I. The fusion protein inserted into membranes from the endoplasmatic reticulum (ER), as confirmed by differential centrifugation and Western analysis. Membrane vesicles containing ProtA–NuoL_N catalyzed the uptake of Na⁺ and K⁺ at rates which were significantly higher than uptake by the control vesicles under identical conditions, demonstrating that ProtA–NuoL_N translocated Na⁺ and K⁺ independently from other complex I subunits. Na⁺ transport by ProtA–NuoL_N was inhibited by EIPA (5-(N-ethyl-N-isopropyl)-amiloride) which specifically reacts with Na⁺/H⁺ antiporters. The cation selectivity and function of the NuoL subunit as a transporter module of the NADH dehydrogenase complex is discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Staphylococcus aureus NuoL-Like Protein MpsA Contributes to the Generation of Membrane Potential

In aerobic microorganisms, the entry point of respiratory electron transfer is represented by the NADH:quinone oxidoreductase. The enzyme couples the oxidation of NADH with the reduction of quinone. In the type 1 NADH:quinone oxidoreductase (Ndh1), this reaction is accompanied by the translocation of cations, such as H⁺ or Na⁺. In Escherichia coli, cation translocation is accomplished by the subunit NuoL, thus generating membrane potential (Δψ). Some microorganisms achieve NADH oxidation by the alternative, nonelectrogenic type 2 NADH:quinone oxidoreductase (Ndh2), which is not cation translocating. Since these enzymes had not been described in Staphylococcus aureus, the goal of this study was to identify proteins operating in the NADH:quinone segment of its respiratory chain. We demonstrated that Ndh2 represents a NADH:quinone oxidoreductase in S. aureus. Additionally, we identified a hypothetical protein in S. aureus showing sequence similarity to the proton-translocating subunit NuoL of complex I in E. coli: the NuoL-like protein MpsA. Mutants with deletion of the nuoL-like gene mpsA and its corresponding operon, mpsABC (mps for membrane potential-generating system), exhibited a small-colony-variant-like phenotype and were severely affected in Δψ and oxygen consumption rates. The MpsABC proteins did not confer NADH oxidation activity. Using an Na⁺/H⁺ antiporter-deficient E. coli strain, we could show that MpsABC constitute a cation-translocating system capable of Na⁺ transport. Our study demonstrates that MpsABC represent an important functional system of the respiratory chain of S. aureus that acts as an electrogenic unit responsible for the generation of Δψ.     

The Na+-Translocating NADH:quinone Oxidoreductase (NDH I) from Klebsiella pneumoniae and Escherichia coli: Implications for the Mechanism of Redox-Driven Cation Translocation by Complex I

Eukaryotic complex I integrated into the respiratory chain transports at least 4 H⁺ per NADH oxidized. Recent results indicate that the cation selectivity is altered to Na⁺ in complex I (NDH I) isolated from the enterobacteria Escherichia coli and Klebsiella pneumoniae. A sequence analysis illustrates the characteristic differences of the enterobacterial, Na⁺-translocating NDH I compared to the H⁺-translocating complex I from mitochondria. Special attention is given to the membranous NuoL (ND5, Nqo12) subunits that possess striking sequence similarities to secondary Na⁺/H⁺ antiporters and are proposed to participate in Na⁺ transport. A model of redox-linked Na⁺ (or H⁺) transport by complex I is discussed based on the ion-pair formation of a negatively charged ubisemiquinone anion with a positively charged Na⁺ (or H⁺).     

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.     

A new hypothesis on the simultaneous direct and indirect proton pump mechanisms in NADH-quinone oxidoreductase (complex I)

Recently, Sazanov’s group reported the X-ray structure of whole complex I [Nature, 465, 441 (2010)], which presented a strong clue for a “piston-like” structure as a key element in an “indirect” proton pump. We have studied the NuoL subunit which has a high sequence similarity to Na⁺/H⁺ antiporters, as do the NuoM and N subunits. We constructed 27 site-directed NuoL mutants. Our data suggest that the H⁺/e⁻ stoichiometry seems to have decreased from (4H⁺/2e⁻) in the wild-type to approximately (3H⁺/2e⁻) in NuoL mutants. We propose a revised hypothesis that each of the “direct” and the “indirect” proton pumps transports 2H⁺ per 2e⁻.     

The novel NhaE-type Na+/H+ antiporter of the pathogenic bacterium Neisseria meningitidis

Neisseria meningitidis is a pathogenic bacterium responsible for meningitis. The mechanisms underlying the control of Na⁺ transmembrane movement, presumably important to pathogenicity, have been barely addressed. To elucidate the function of the components of the Na⁺ transport system in N. meningitidis, an open reading frame from the genome of this bacterium displaying similarity with the NhaE type of Na⁺/H⁺ antiporters was expressed in Escherichia coli and characterized for sodium transport ability. The N. meningitidis antiporter (NmNhaE) was able to complement an E. coli strain devoid of Na⁺/H⁺ antiporters (KNabc) respecting the ability to grow in the presence of NaCl and LiCl. Ion transport assays in everted vesicles prepared from KNabc expressing NmNhaE from a plasmid confirmed its ability to translocate Na⁺ and Li⁺. Here is presented the characterization of the first NhaE from a pathogen, an important contribution to the comprehension of sodium ion metabolism in this kind of microorganisms.     

The Deactive Form of Respiratory Complex I from Mammalian Mitochondria Is a Na+/H+ Antiporter

In mitochondria, complex I (NADH:ubiquinone oxidoreductase) uses the redox potential energy from NADH oxidation by ubiquinone to transport protons across the inner membrane, contributing to the proton-motive force. However, in some prokaryotes, complex I may transport sodium ions instead, and three subunits in the membrane domain of complex I are closely related to subunits from the Mrp family of Na⁺/H⁺ antiporters. Here, we define the relationship between complex I from Bos taurus heart mitochondria, a close model for the human enzyme, and sodium ion transport across the mitochondrial inner membrane. In accord with current consensus, we exclude the possibility of redox-coupled Na⁺ transport by B. taurus complex I. Instead, we show that the “deactive” form of complex I, which is formed spontaneously when enzyme turnover is precluded by lack of substrates, is a Na⁺/H⁺ antiporter. The antiporter activity is abolished upon reactivation by the addition of substrates and by the complex I inhibitor rotenone. It is specific for Na⁺ over K⁺, and it is not exhibited by complex I from the yeast Yarrowia lipolytica, which thus has a less extensive deactive transition. We propose that the functional connection between the redox and transporter modules of complex I is broken in the deactive state, allowing the transport module to assert its independent properties. The deactive state of complex I is formed during hypoxia, when respiratory chain turnover is slowed, and may contribute to determining the outcome of ischemia-reperfusion injury.     

In?Vitro Demonstration of Dual Light-Driven Na+/H+ Pumping by a Microbial Rhodopsin

A subfamily of rhodopsin pigments was recently discovered in bacteria and proposed to function as dual-function light-driven H⁺/Na⁺ pumps, ejecting sodium ions from cells in the presence of sodium and protons in its absence. This proposal was based primarily on light-induced proton flux measurements in suspensions of Escherichia coli cells expressing the pigments. However, because E. coli cells contain numerous proteins that mediate proton fluxes, indirect effects on proton movements involving endogenous bioenergetics components could not be excluded. Therefore, an in vitro system consisting of the purified pigment in the absence of other proteins was needed to assign the putative Na⁺ and H⁺ transport definitively. We expressed IAR, an uncharacterized member from Indibacter alkaliphilus in E. coli cell suspensions, and observed similar ion fluxes as reported for KR2 from Dokdonia eikasta. We purified and reconstituted IAR into large unilamellar vesicles (LUVs), and demonstrated the proton flux criteria of light-dependent electrogenic Na⁺ pumping activity in vitro, namely, light-induced passive proton flux enhanced by protonophore. The proton flux was out of the LUV lumen, increasing lumenal pH. In contrast, illumination of the LUVs in a Na⁺-free suspension medium caused a decrease of lumenal pH, eliminated by protonophore. These results meet the criteria for electrogenic Na⁺ transport and electrogenic H⁺ transport, respectively, in the presence and absence of Na⁺. The direction of proton fluxes indicated that IAR was inserted inside-out into our sealed LUV system, which we confirmed by site-directed spin-label electron paramagnetic resonance spectroscopy. We further demonstrate that Na⁺ transport by IAR requires Na⁺ only on the cytoplasmic side of the protein. The in vitro LUV system proves that the dual light-driven H⁺/Na⁺ pumping function of IAR is intrinsic to the single rhodopsin protein and enables study of the transport activities without perturbation by bioenergetics ion fluxes encountered in vivo.     

Keeping It Simple,Transport Mechanism and pH Regulation in Na+/H+ Exchangers

Na⁺/H⁺ exchangers are essential for regulation of intracellular proton and sodium concentrations in all living organisms. We examined and experimentally verified a kinetic model for Na⁺/H⁺ exchangers, where a single binding site is alternatively occupied by Na⁺ or one or two H⁺ ions. The proposed transport mechanism inherently down-regulates Na⁺/H⁺ exchangers at extreme pH, preventing excessive cytoplasmic acidification or alkalinization. As an experimental test system we present the first electrophysiological investigation of an electroneutral Na⁺/H⁺ exchanger, NhaP1 from Methanocaldococcus jannaschii (MjNhaP1), a close homologue of the medically important eukaryotic NHE Na⁺/H⁺ exchangers. The kinetic model describes the experimentally observed substrate dependences of MjNhaP1, and the transport mechanism explains alkaline down-regulation of MjNhaP1. Because this model also accounts for acidic down-regulation of the electrogenic NhaA Na⁺/H⁺ exchanger from Escherichia coli (EcNhaA, shown in a previous publication) we conclude that it applies generally to all Na⁺/H⁺ exchangers, electrogenic as well as electroneutral, and elegantly explains their pH regulation. Furthermore, the electrophysiological analysis allows insight into the electrostatic structure of the translocation complex in electroneutral and electrogenic Na⁺/H⁺ exchangers.     

Single Site Mutations in the Hetero-oligomeric Mrp Antiporter from Alkaliphilic Bacillus pseudofirmus OF4 That Affect Na+/H+ Antiport Activity,Sodium Exclusion,Individual Mrp Protein Levels,or Mrp Complex Formation

Mrp systems are widely distributed and structurally complex cation/proton antiporters. Antiport activity requires hetero-oligomeric complexes of all six or seven hydrophobic Mrp proteins (MrpA–MrpG). Here, a panel of site-directed mutants in conserved or proposed motif residues was made in the Mrp Na⁺(Li⁺)/H⁺ antiporter from an alkaliphilic Bacillus. The mutant operons were expressed in antiporter-deficient Escherichia coli KNabc and assessed for antiport properties, support of sodium resistance, membrane levels of each Mrp protein, and presence of monomeric and dimeric Mrp complexes. Antiport did not depend on a VFF motif or a conserved tyrosine pair, but a role for a conserved histidine in a potential quinone binding site of MrpA was supported. The importance of several acidic residues for antiport was confirmed, and the importance of additional residues was demonstrated (e.g. three lysine residues conserved across MrpA, MrpD, and membrane-bound respiratory Complex I subunits (NuoL/M/N)). The results extended indications that MrpE is required for normal membrane levels of other Mrp proteins and for complex formation. Moreover, mutations in several other Mrp proteins lead to greatly reduced membrane levels of MrpE. Thus, changes in either of the two Mrp modules, MrpA–MrpD and MrpE–MrpG, influence the other. Two mutants, MrpB-P37G and MrpC-Q70A, showed a normal phenotype but lacked the MrpA–MrpG monomeric complex while retaining the dimeric hetero-oligomeric complex. Finally, MrpG-P81A and MrpG-P81G mutants exhibited no antiport activity but supported sodium resistance and a low [Na⁺]_in. Such mutants could be used to screen hypothesized but uncharacterized sodium efflux functions of Mrp apart from Na⁺ (Li⁺)/H⁺ antiport.     

Membrane Na+-pyrophosphatases Can Transport Protons at Low Sodium Concentrations

Membrane-bound Na⁺-pyrophosphatase (Na⁺-PPase), working in parallel with the corresponding ATP-energized pumps, catalyzes active Na⁺ transport in bacteria and archaea. Each ∼75-kDa subunit of homodimeric Na⁺-PPase forms an unusual funnel-like structure with a catalytic site in the cytoplasmic part and a hydrophilic gated channel in the membrane. Here, we show that at subphysiological Na⁺ concentrations (<5 mm), the Na⁺-PPases of Chlorobium limicola, four other bacteria, and one archaeon additionally exhibit an H⁺-pumping activity in inverted membrane vesicles prepared from recombinant Escherichia coli strains. H⁺ accumulation in vesicles was measured with fluorescent pH indicators. At pH 6.2–8.2, H⁺ transport activity was high at 0.1 mm Na⁺ but decreased progressively with increasing Na⁺ concentrations until virtually disappearing at 5 mm Na⁺. In contrast, ²²Na⁺ transport activity changed little over a Na⁺ concentration range of 0.05–10 mm. Conservative substitutions of gate Glu²⁴² and nearby Ser²⁴³ and Asn⁶⁷⁷ residues reduced the catalytic and transport functions of the enzyme but did not affect the Na⁺ dependence of H⁺ transport, whereas a Lys⁶⁸¹ substitution abolished H⁺ (but not Na⁺) transport. All four substitutions markedly decreased PPase affinity for the activating Na⁺ ion. These results are interpreted in terms of a model that assumes the presence of two Na⁺-binding sites in the channel: one associated with the gate and controlling all enzyme activities and the other located at a distance and controlling only H⁺ transport activity. The inherent H⁺ transport activity of Na⁺-PPase provides a rationale for its easy evolution toward specific H⁺ transport.     

Functional role of the MrpA- and MrpD-homologous protein subunits in enzyme complexes evolutionary related to respiratory chain complex I

NADH:quinone oxidoreductase or complex I is a large membrane bound enzyme complex that has evolved from the combination of smaller functional building blocks. Intermediate size enzyme complexes exist in nature that comprise some, but not all of the protein subunits in full size 14-subunit complex I. The membrane spanning complex I subunits NuoL, NuoM and NuoN are homologous to each other and to two proteins from one particular class of Na⁺/H⁺ antiporters, denoted MrpA and MrpD. In complex I, these ion transporter protein subunits are prime candidates for harboring important parts of the proton pumping machinery. Using a model system, consisting of Bacillus subtilis MrpA and MrpD deletion strains and a low copy expression plasmid, it was recently demonstrated that NuoN can rescue the strain deleted for MrpD but not that deleted for MrpA, whereas the opposite tendency was seen for NuoL. This demonstrated that the MrpA-type and MrpD-type proteins have unique functional specializations. In this work, the corresponding antiporter-like protein subunits from the smaller enzymes evolutionarily related to complex I were tested in the same model system. The subunits from 11-subunit complex I from Bacillus cereus behaved essentially as those from full size complex I, corroborating that this enzyme should be regarded as a bona fide complex I. The hydrogenase-3 and hydrogenase-4 antiporter-like proteins on the other hand, could substitute equally well for MrpA or MrpD at pH 7.4, suggesting that these enzymes have intermediate forms of the antiporter-like proteins, which seemingly lack the functional specificity.     

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.     

Bacterial transporters: Charge translocation and mechanism

A comparative review of the electrophysiological characterization of selected secondary active transporters from Escherichia coli is presented. In melibiose permease MelB and the Na⁺/proline carrier PutP pre-steady-state charge displacements can be assigned to an electrogenic conformational transition associated with the substrate release process. In both transporters cytoplasmic release of the sugar or the amino acid as well as release of the coupling cation are associated with a charge displacement. This suggests a common transport mechanism for both transporters. In the NhaA Na⁺/H⁺ exchanger charge translocation due to its steady-state transport activity is observed. A new model is proposed for pH regulation of NhaA that is based on coupled Na⁺ and H⁺ equilibrium binding.     

Metagenomic cloning and characterization of Na+/H+ antiporter genes taken from sediments in Chaerhan Salt Lake in China

A metagenomic library containing 8,000 clones was constructed by using genomic DNA obtained from Chaerhan Salt Lake in northwest China. Three Na⁺/H⁺ antiporters, C4-NhaG, C47-NhaG and C49-NhaG that grouped to the NhaG family, were screened and cloned from this metagenome by complementing Escherichia coli strain KNabc (ΔnhaA ΔnhaB ΔchaA) in medium containing 0.2 M NaCl. The three putative Na⁺/H⁺ antiporters were membrane proteins with 10, 11 and 11 transmembrane segments, respectively. They enabled E. coli KNabc to grow in medium containing 0.2–0.6 M Na⁺ or 7–14 mM Li⁺. Everted membrane vesicles prepared from E. coli KNabc cells carrying C49-NhaG exhibited Na⁺/H⁺ and Li⁺/H⁺ antiport activities.     

Cloning and identification of a novel NhaD-type Na+/H+ antiporter from metagenomic DNA of the halophilic bacteria in soil samples around Daban Salt Lake

In this study, metagenomic DNA was screened for the Na⁺/H⁺ antiporter gene from the halophilic bacteria in Daban Salt Lake by selection in Escherichia coli KNabc lacking three major Na⁺/H⁺ antiporters. One gene designated as Hb_nhaD encoding a novel NhaD-type Na⁺/H⁺ antiporter was finally cloned. The presence of Hb_NhaD conferred tolerance of E. coli KNabc to up to 0.5 M NaCl and 0.2 M LiCl, and an alkaline pH. Hb_NhaD has the highest identity (70.6 %) with a putative NhaD-type Na⁺/H⁺ antiporter from an uncharacterized Clostridiaceae species, and also has lower identity with known NhaD-type Na⁺/H⁺ antiporters from Halomonas elongata (20.8 %), Alkalimonas amylolytica (19.0 %), Vibrio parahaemolyticus (18.9 %) and Vibrio cholerae (18.7 %). pH-dependent Na⁺(Li⁺)/H⁺ antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc carrying Hb_nhaD. Hb_NhaD exhibited very high Na⁺(Li⁺)/H⁺ antiport activity over a wide pH range from 6.5 to 9.0 with the highest activity at pH 7.0 which is significantly different from those of the above known NhaD-type Na⁺/H⁺ antiporters. Also, the apparent K _m values of Hb_NhaD for Na⁺ and Li⁺ at pH 7.0 were determined to be 1.31 and 2.16, respectively. Based on the above results, we proposed that Hb_NhaD should be categorized as a novel NhaD-type Na⁺/H⁺ antiporter.     

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.     

A novel method to quantify H-ATPase-dependent Na transport across plasma membrane vesicles

To prevent sodium toxicity in plants, Na⁺ is excluded from the cytosol to the apoplast or the vacuole by Na⁺/H⁺ antiporters. The secondary active transport of Na⁺ to apoplast against its electrochemical gradient is driven by plasma membrane H⁺-ATPases that hydrolyze ATP and pump H⁺ across the plasma membrane. Current methods to determine Na⁺ flux rely either on the use of Na-isotopes (²²Na) which require special working permission or sophisticated equipment or on indirect methods estimating changes in the H⁺ gradient due to H⁺-ATPase in the presence or absence of Na⁺ by pH-sensitive probes. To date, there are no methods that can directly quantify H⁺-ATPase-dependent Na⁺ transport in plasma membrane vesicles. We developed a method to measure bidirectional H⁺-ATPase-dependent Na⁺ transport in isolated membrane vesicle systems using atomic absorption spectrometry (AAS). The experiments were performed using plasma membrane-enriched vesicles isolated by aqueous two-phase partitioning from leaves of Populus tomentosa. Since most of the plasma membrane vesicles have a sealed right-side-out orientation after repeated aqueous two-phase partitioning, the ATP-binding sites of H⁺-ATPases are exposed towards inner side. Leaky vesicles were preloaded with Na⁺ sealed for the study of H⁺-ATPase-dependent Na⁺ transport. Our data implicate that Na⁺ movement across vesicle membranes is highly dependent on H⁺-ATPase activity requiring ATP and Mg²⁺ and displays optimum rates of 2.50 μM Na⁺ mg^− 1 membrane protein min^− 1 at pH 6.5 and 25 °C. In this study, for the first time, we establish new protocols for the preparation of sealed preloaded right-side-out vesicles for the study of H⁺-ATPase-dependent Na⁺ transport. The results demonstrate that the Na⁺ content of various types of plasma membrane vesicle can be directly quantified by AAS, and the results measured using AAS method were consistent with those determined by the previous established fluorescence probe method. The method is a convenient system for the study of bidirectional H⁺-ATPase-dependent Na⁺ transport with membrane vesicles.     

Nitric Oxide Mediates Root K+/Na+ Balance in a Mangrove Plant,Kandelia obovata,by Enhancing the Expression of AKT1-Type K+ Channel and Na+/H+ Antiporter under High Salinity

It is well known that nitric oxide (NO) enhances salt tolerance of glycophytes. However, the effect of NO on modulating ionic balance in halophytes is not very clear. This study focuses on the role of NO in mediating K⁺/Na⁺ balance in a mangrove species, Kandelia obovata Sheue, Liu and Yong. We first analyzed the effects of sodium nitroprusside (SNP), an NO donor, on ion content and ion flux in the roots of K. obovata under high salinity. The results showed that 100 μM SNP significantly increased K⁺ content and Na⁺ efflux, but decreased Na⁺ content and K⁺ efflux. These effects of NO were reversed by specific NO synthesis inhibitor and scavenger, which confirmed the role of NO in retaining K⁺ and reducing Na⁺ in K. obovata roots. Using western-blot analysis, we found that NO increased the protein expression of plasma membrane (PM) H⁺-ATPase and vacuolar Na⁺/H⁺ antiporter, which were crucial proteins for ionic balance. To further clarify the molecular mechanism of NO-modulated K⁺/Na⁺ balance, partial cDNA fragments of inward-rectifying K⁺ channel, PM Na⁺/H⁺ antiporter, PM H⁺-ATPase, vacuolar Na⁺/H⁺ antiporter and vacuolar H⁺-ATPase subunit c were isolated. Results of quantitative real-time PCR showed that NO increased the relative expression levels of these genes, while this increase was blocked by NO synthesis inhibitors and scavenger. Above results indicate that NO greatly contribute to K⁺/Na⁺ balance in high salinity-treated K. obovata roots, by activating AKT1-type K⁺ channel and Na⁺/H⁺ antiporter, which are the critical components in K⁺/Na⁺ transport system.