2-Aminoperimidine, a specific inhibitor of bacterial NhaA Na(+)/H(+) antiporters

The diuretic drug amiloride and its numerous derivatives are competitive inhibitors of mammalian Na(+)/H(+) antiporters and other eukaryotic antiporters. Most prokaryotic antiporters, including the major NhaA family of enterobacteria, are resistant to these compounds. We show that 2-aminoperimidine (AP), a guanidine-containing naphthalene derivative with some similarity to amiloride, acts as a specific inhibitor of NhaA from Escherichia coli. Similar concentrations (IC(50) of 0.9 muM) inhibit the proton motive force dependent Na(+)(Li(+))/H(+) exchange reaction in inside-out sub-bacterial vesicles (at 10 mM NaCl, pH 8) as well as the initial rate of (22)Na(+)/Na(+) exchange mediated by pure NhaA in proteoliposomes. The inhibitor is specific to NhaA type antiporters, so AP is a new tool to study the mechanism and roles of NhaA antiporters of enterobacteria as well as the molecular basis of inhibition by an amiloride-like compound.     

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.     

Na(+)/H(+) exchange in mosquito Malpighian tubules

Fluid secretion and intracellular pH were measured in isolated mosquito Malpighian tubules to determine the presence of Na(+)/H(+) exchange. Rates of fluid secretion by individual Malpighian tubules in vitro were inhibited by 78% of control in the presence of 100 microM 5-(N-ethyl-n-isopropyl)-amiloride (EIPA), a specific inhibitor of Na(+)/H(+) exchange. Steady-state intracellular pH was measured microfluorometrically by using 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in individual Malpighian tubules. Bathing the Malpighian tubules in 0 mM extracellular Na(+) or in the presence of 100 microM EIPA reduced the steady-state intracellular pH by 0.5 pH units. Stimulation of the Na(+)/H(+) exchanger by using the NH(4)Cl pulse technique resulted in a rate of recovery from the NH(4)Cl-induced acute acid load of 8.7 +/- 1.0 x 10(-3) pH/s. The rates of recovery of intracellular pH after the acute acid load in the absence of extracellular Na(+) or in the presence of 100 microM EIPA were 0.7 +/- 0.6 and -0.3 +/- 0.3 x 10(-3) pH/s, respectively. These results indicate that mosquito Malpighian tubules possess a Na(+)/H(+) exchanger.     

Functional expression in Escherichia coli of low-affinity and high-affinity Na(+)(Li(+))/H(+) antiporters of Synechocystis

Synechocystis sp. strain PCC 6803 has five genes for putative Na(+)/H(+) antiporters (designated nhaS1, nhaS2, nhaS3, nhaS4, and nhaS5). The deduced amino acid sequences of NhaS1 and NhaS2 are similar to that of NhaP, the Na(+)/H(+) antiporter of Pseudomonas aeruginosa, whereas those of NhaS3, NhaS4, and NhaS5 resemble that of NapA, the Na(+)/H(+) antiporter of Enterococcus hirae. We successfully induced the expression of nhaS1, nhaS3, and nhaS4 under control of an Na(+)-dependent promoter in Escherichia coli TO114, a strain that is deficient in Na(+)/H(+) antiport activity. Inverted membrane vesicles prepared from TO114 nhaS1 and TO114 nhaS3 cells exhibited Na(+)(Li(+))/H(+) antiport activity. Kinetic analysis of this activity revealed that nhaS1 encodes a low-affinity Na(+)/H(+) antiporter with a K(m) of 7.7 mM for Na(+) ions and a K(m) of 2.5 mM for Li(+) ions, while nhaS3 encodes a high-affinity Na(+)/H(+) antiporter with a K(m) of 0.7 mM for Na(+) ions and a K(m) of 0.01 mM for Li(+) ions. Transformation of E. coli TO114 with the nhaS1 and nhaS3 genes increased cellular tolerance to high concentrations of Na(+) and Li(+) ions, as well as to depletion of K(+) ions during cell growth. To our knowledge, this is the first functional characterization of Na(+)/H(+) antiporters from a cyanobacterium. Inverted membrane vesicles prepared from TO114 nhaS4 cells did not have Na(+)/H(+) antiport activity, and the cells themselves were as sensitive to Na(+) and Li(+) ions as the original TO114 cells. However, the TO114 nhaS4 cells were tolerant to depletion of K(+) ions. Taking into account these results and the growth characteristics of Synechocystis mutants in which nhaS genes had been inactivated by targeted disruption, we discuss possible roles of NhaS1, NhaS3, and NhaS4 in Synechocystis.     

Insect Na(+)/K(+)-ATPase

Na(+)/K(+)-ATPase (sodium/potassium pump) is a P-type ion-motive ATPase found in the plasma membranes of animal cels. In vertebrates, the functions of this enzyme in nerves, heart and kidney are well characterized and characteristics a defined by different isoforms. In contrast, despite different tissue distributions, insects possess a single isoform of the alpha-subunit. A comparison of insect and vertebrate Na(+)/K(+)-ATPases reveals that although the mode of action and structure are very highly conserved, the specific roles of the enzyme in most tissues varies. However, the enzyme is essential for the function of nerve cells, and in this respect Na(+)/K(+)-ATPase appears to be fundamental in metazoan evolution.     

Na+/H+ antiporters

Na+/H+ antiports or exchange reactions have been found widely, if not ubiquitously, in prokaryotic and eukaryotic membranes. In any given experimental system, the multiplicity of ion conductance pathways and the absence of specific inhibitors complicate efforts to establish that the antiport observed actually results from the activity of a specific secondary porter which catalyzes coupled exchanged of the two ions. Nevertheless, a large body of evidence suggests that at least some prokaryotes possess a delta psi-dependent, mutable Na+/H+ antiporter which catalyzes Na+ extrusion in exchange for H+; in other bacterial species, the antiporter my function electroneutrally, at least at some external pH values. The bacterial Na+/H+ antiporter constitutes a critical limb of Na+ circulation, functioning to maintain a delta mu Na+ for use by Na+-coupled bioenergetic processes. The prokaryotic antiporter is also involved in pH homeostasis in the alkaline pH range. Studies of mutant strains that are deficient in Na+/H+ antiporter activity also indicate the existence of a relationship, e.g., a common subunit or regulatory factor, between the Na+/H+ antiporter and Na+/solute symporters in several bacterial species. In eukaryotes, an electroneutral, amiloride-sensitive Na+/H+ antiport has been found in a wide variety of cell and tissue types. Generally, the normal direction of the antiport appears to be that of Na+ uptake and H+ extrusion. The activity is thus implicated as part of a complex system for Na+ circulation, e.g., in transepithelial transport, and might have some role in acidification in the renal proximal tubule. In many experimental systems, the Na+/H+ antiport appears to influence intracellular pH. In addition to a role in general pH homeostasis, such Na+-dependent changes in intracellular pH could be part of the early events in a variety of differentiating and proliferative systems. Reconstitution and structural studies, as well as detailed analysis of gene loci and products which affect the antiport activity, are in their very early stages. These studies will be important in further clarification of the precise structural nature and role(s) of the Na+/H+ antiporters. In neither prokaryotes nor eukaryotes systems is there yet incontrovertible evidence that a specific protein carrier, that catalyzes Na+/H+ antiport, is actually responsible for any of the multitude of effects attributed to such antiporters. The Na+-H+ exchange might turn out to be side reactions of other porters or the additive effects of several conductance pathways; or, as appears most likely in at least some bacteria and in renal tissue, the antiporter may be a discrete, complex carr     

The endosomal Na(+)/H(+) exchanger contributes to multivesicular body formation by regulating the recruitment of ESCRT-0 Vps27p to the endosomal membrane

Multivesicular bodies (MVBs) are late endosomal compartments containing luminal vesicles (MVB vesicles) that are formed by inward budding of the endosomal membrane. In budding yeast, MVBs are an important cellular mechanism for the transport of membrane proteins to the vacuolar lumen. This process requires a class E subset of vacuolar protein sorting (VPS) genes. VPS44 (allelic to NHX1) encodes an endosome-localized Na(+)/H(+) exchanger. The function of the VPS44 exchanger in the context of vacuolar protein transport is largely unknown. Using a cell-free MVB formation assay system, we demonstrated that Nhx1p is required for the efficient formation of MVB vesicles in the late endosome. The recruitment of Vps27p, a class E Vps protein, to the endosomal membrane was dependent on Nhx1p activity and was enhanced by an acidic pH at the endosomal surface. Taken together, we propose that Nhx1p contributes to MVB formation by the recruitment of Vps27p to the endosomal membrane, possibly through Nhx1p antiporter activity.     

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.     

The putative plasma membrane Na(+)/H(+) antiporter SOS1 controls long-distance Na(+) transport in plants

The salt tolerance locus SOS1 from Arabidopsis has been shown to encode a putative plasma membrane Na(+)/H(+) antiporter. In this study, we examined the tissue-specific pattern of gene expression as well as the Na(+) transport activity and subcellular localization of SOS1. When expressed in a yeast mutant deficient in endogenous Na(+) transporters, SOS1 was able to reduce Na(+) accumulation and improve salt tolerance of the mutant cells. Confocal imaging of a SOS1-green fluorescent protein fusion protein in transgenic Arabidopsis plants indicated that SOS1 is localized in the plasma membrane. Analysis of SOS1 promoter-beta-glucuronidase transgenic Arabidopsis plants revealed preferential expression of SOS1 in epidermal cells at the root tip and in parenchyma cells at the xylem/symplast boundary of roots, stems, and leaves. Under mild salt stress (25 mM NaCl), sos1 mutant shoot accumulated less Na(+) than did the wild-type shoot. However, under severe salt stress (100 mM NaCl), sos1 mutant plants accumulated more Na(+) than did the wild type. There also was greater Na(+) content in the xylem sap of sos1 mutant plants exposed to 100 mM NaCl. These results suggest that SOS1 is critical for controlling long-distance Na(+) transport from root to shoot. We present a model in which SOS1 functions in retrieving Na(+) from the xylem stream under severe salt stress, whereas under mild salt stress it may function in loading Na(+) into the xylem.     

Altered cardiac Na(+)/H(+) exchange in phospholipase D-treated sarcolemmal vesicles

Cardiac sarcolemmal Na(+)/H(+) exchange is critical for the regulation of intracellular pH, and its activity contributes to ischemia-reperfusion injury. It has been suggested that the membrane phospholipid environment does not modulate Na(+)/H(+) exchange. The present study was carried out to determine the effects on Na(+)/H(+) exchange of modifying the endogenous membrane phospholipids through the addition of exogenous phospholipase D. Incubation of 0.825 U of phospholipase D with 1 mg of porcine cardiac sarcolemmal vesicles hydrolyzed 34 +/- 2% of the sarcolemmal phosphatidylcholine and increased phosphatidic acid 10.2 +/- 0.5-fold. Treatment of vesicles with phospholipase D resulted in a 46 +/- 2% inhibition of Na(+)/H(+) exchange. Na(+)/H(+) exchange was measured as a function of reaction time, extravesicular pH, and extravesicular Na(+). All of these parameters of Na(+)/H(+) exchange were inhibited following phospholipase D treatment compared with untreated controls. Passive efflux of Na(+) was unaffected. Treatment of sarcolemmal vesicles with phospholipase C had no effect on Na(+)/H(+) exchange. We conclude that phospholipase D-induced changes in the cardiac sarcolemmal membrane phospholipid environment alter Na(+)/H(+) exchange.     

Rapamycin sensitive ROS formation and Na(+)/H(+) exchanger activity in dendritic cells

Rapamycin, a widely used immunosuppressive drug, has been shown to interfere with the function of dendritic cells (DCs), antigen-presenting cells contributing to the initiation of primary immune responses and the establishment of immunological memory. DC function is governed by the Na(+)/H(+) exchanger (NHE), which is activated by bacterial lipopolysaccharides (LPS) and is required for LPS-induced cell swelling, reactive oxygen species (ROS) production and TNF-α release. The present study explored, whether rapamycin influences NHE activity and/or ROS formation in DCs. Mouse DCs were treated with LPS in the absence and presence of rapamycin (100 nM). ROS production was determined from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, cytosolic pH (pH(i)) from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, and TNF-α production utilizing ELISA. In the absence of LPS, rapamycin did not significantly modify cytosolic pH, NHE activity or cell volume but significantly decreased ROS formation. LPS stimulated NHE activity, enhanced forward scatter, increased ROS formation, and triggered TNF-α release, effects all blunted in the presence of rapamycin. NADPH oxidase inhibitor Vas-2870 (10 μM) mimicked the effect of rapamycin on LPS induced stimulation of NHE activity and TNF-α release. The effect of rapamycin on TNF-α release was also mimicked by the antioxidant ROS scavenger Tempol (30 μM) and partially reversed by additional application of tert-butylhydroperoxide (10 μM). In conclusion, in DCs rapamycin disrupts LPS induced ROS formation with subsequent inhibition of NHE activity, cell swelling and TNF-α release.     

Identification of a pH regulated Na(+)/H(+) antiporter of Methanococcus jannaschii

The genome of the hyperthermophilic archaeon Methanococcus jannaschii contains three Na(+)/H(+) antiporter related genes Mj0057, Mj1521 and Mj1275. Comparative sequence alignments revealed that Mj0057 and Mj1521 belong to the NhaP family whereas Mj1275 is a member of the NapA family. The genes were cloned and expressed in the double mutant Escherichia coli strain Frag144 (DeltanhaA, DeltanhaB) to analyze their capability of mediating DeltapH driven Na(+) flux in everted vesicles. From the tested clones only Mj0057 displayed Na(+) (Li(+))/H(+) antiporter activity. The transport was pH dependent and occurred at pH 7.0 and below. At pH 6.0 the apparent K(m) values for Na(+) and Li(+) were approximately 10 and 2.5 mM, respectively.     

Intracellular pH regulation by Na(+)/H(+) exchange requires phosphatidylinositol 4,5-bisphosphate

The carrier-mediated, electroneutral exchange of Na(+) for H(+) across the plasma membrane does not directly consume metabolic energy. Nevertheless, acute depletion of cellular ATP markedly decreases transport. We analyzed the possible involvement of polyphosphoinositides in the metabolic regulation of NHE1, the ubiquitous isoform of the Na(+)/H(+) exchanger. Depletion of ATP was accompanied by a marked reduction of plasmalemmal phosphatidylinositol 4,5-bisphosphate (PIP(2)) content. Moreover, sequestration or hydrolysis of plasmalemmal PIP(2), in the absence of ATP depletion, was associated with profound inhibition of NHE1 activity. Examination of the primary structure of the COOH-terminal domain of NHE1 revealed two potential PIP(2)-binding motifs. Fusion proteins encoding these motifs bound PIP(2) in vitro. When transfected into antiport-deficient cells, mutant forms of NHE1 lacking the putative PIP(2)-binding domains had greatly reduced transport capability, implying that association with PIP(2) is required for optimal activity. These findings suggest that NHE1 activity is modulated by phosphoinositides and that the inhibitory effect of ATP depletion may be attributable, at least in part, to the accompanying net dephosphorylation of PIP(2).     

Effect of azathioprine on Na(+)/H(+) exchanger activity in dendritic cells

Azathioprine is a powerful immunosuppressive drug, which is partially effective by interfering with the maturation and function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs are stimulated by bacterial lipopolysaccharides (LPS), which trigger the formation of reactive oxygen species (ROS), paralleled by activation of the Na(+)/H(+) exchanger. The carrier is involved in the regulation of cytosolic pH, cell volume and migration. The present study explored whether azathioprine influences Na(+)/H(+) exchanger activity in DCs. DCs were isolated from murine bone marrow, cytosolic pH (pH(i)) was estimated utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM) fluorescence, Na(+)/H(+) exchanger activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNFα release utilizing ELISA, and migration utilizing transwell migration assays. Exposure of DCs to lipopolysaccharide (LPS, 1 μg/ml) led to a transient increase of Na(+)/H(+) exchanger activity, an effect paralleled by ROS formation, increased cell volume, TNFα production and stimulated migration. Azathioprine (10 μM) did not significantly alter the Na(+)/H(+) exchanger activity, cell volume and ROS formation prior to LPS exposure but significantly blunted the LPS-induced stimulation of Na(+)/H(+) exchanger activity, ROS formation, cell swelling, TNFα production and cell migration. In conclusion, azathioprine interferes with the activation of dendritic cell Na(+)/H(+) exchanger by bacterial lipopolysaccharides, an effect likely participating in the anti-inflammatory action of the drug.     

Functional expression of plant plasma membrane H(+)-ATPase in yeast endoplasmic reticulum.

Recombinant plant plasma membrane H(+)-ATPase has been produced in a yeast expression system comprising a multicopy plasmid and the strong promoter of the yeast PMA1 gene. Western blotting with a specific monoclonal antibody showed that the plant ATPase is one of the major membrane proteins made by the transformed cells, accounting for about 1% of total yeast protein. The plant ATPase synthesized in yeast is fully active. It hydrolyzes ATP, pumps protons, and the reaction cycle involves a phosphorylated intermediate. Phosphorylation is possible from both ATP and Pi. Unlike the situation in plants, however, most of the plant ATPase is not expressed in the yeast plasma membrane. Rather, the enzyme appears to remain trapped at a very early stage of secretory pathway: insertion into the endoplasmic reticulum. This organelle was observed to proliferate in the form of stacked membranes surrounding the yeast nucleus in order to accommodate the large amount of plant ATPase produced. In this location, the plant ATPase can be purified with high yield (70 mg from 1 kg of yeast) from membranes devoid of endogenous yeast plasma membrane H(+)-ATPase. This convenient expression system could be useful for other eukaryotic membrane proteins and ATPases.