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At least 19 recordsLinked to original sources
PubMed1992

Reconstitution of Na+/H(+)-antiporter of bovine renal brush-border membrane into proteoliposomes and detection of a 110 kDa protein cross-reactive with antibodies against a human Na+/H(+)-antiporter partial peptide in antiport-active fractions after partial fractionation.

Bovine renal brush-border membranes were solubilized by 1.6% sodium cholate. Na+/H(+)-antiporter was recovered in the supernatant after centrifugation at 160,000 x g for 1 h and was successfully reconstituted into proteoliposomes by a cholate-dialysis procedure. The reconstituted Na+/H(+)-antiporter showed a pH-gradient dependent and amiloride-sensitive 22Na+ uptake very similar to that of brush-border membrane vesicles. Factors affecting the efficiency of reconstitution as well as the stability of the solubilized antiporter at various temperatures were studied. Sodium cholate-solubilized brush-border membrane proteins were fractionated by Sephacryl S-400 and DEAE-Toyopearl chromatography, and fractions containing reconstitutively active Na+/H(+)-antiporter were identified. A 110 kDa peptide cross-reactive with a polyclonal antibody against a C-terminal peptide (22-amino acid residues) of human Na+/H(+)-antiporter was consistently found on the immunoblot of the active fractions. A closely similar peptide was also detected in human placental membranes by this antibody. These results strongly suggest that the 110 kDa protein is responsible for Na+/H(+)-antiporter activity.

Animals↗
PubMed1993

Cloning and characterization of a putative Ca2+/H+ antiporter gene from Escherichia coli upon functional complementation of Na+/H+ antiporter-deficient strains by the overexpressed gene.

DNA libraries from alkaliphilic Bacillus firmus OF4 had been screened earlier (Ivey, D.M., Guffanti, A.A., Bossewitch, J. S., Padan, E., and Krulwich, T. A. (1991) J. Biol. Chem. 266, 23483-23489) for clones that would functionally complement a strain of Escherichia coli (NM81) with a deletion in one of its Na+/H+ antiporter genes. During those studies, an alkaliphile antiporter gene was hypothesized to have been incorporated into the chromosome of strain NM81, producing Na(+)-resistant NM8191. After introduction of a deletion in the second known E. coli Na+/H+ antiporter gene, libraries were prepared from NM8191 and screened for complementation of Na+/H+ antiporter-deficient mutants of E. coli. Instead of retrieving an alkaliphile gene, an unexpected E. coli gene was identified on the basis of its ability to restore Na+ resistance and membrane Na+/H+ antiporter activity to such mutant strains. The active open reading frame in the clone maps at 27 min on the E. coli chromosome and is identical in sequence to a wild type counterpart. It would be predicted to encode an extremely hydrophobic protein with multiple membrane-spanning regions and a molecular weight of 39,200. A region in one of the predicted hydrophilic loops in the gene product structure possesses striking sequence similarity to calsequestrin. The Ca2+/H+ antiporter activity of membranes from an E. coli transformant with a clone possessing only this open reading frame was indeed found to have enhanced pH-independent Ca2+/H+ antiporter activity. The Ca2+/H+ and Na+/H+ antiporter activities conferred by the clone were both inhibited by Mg2+. The gene was designated chaA and is proposed to be the structural gene for a Ca2+/H+ antiporter whose overexpression leads to resistance to growth inhibition by both calcium and sodium.

Amino Acid Sequence↗
PubMed1986

Functional expression of a human Na+/H+ antiporter gene transfected into antiporter-deficient mouse L cells.

To clone the gene for the human Na+/H+ antiporter, we first constructed a stable mouse LTK- cell line (LAP1) lacking Na+/H+ antiport activity. Second, we devised a selective technique based on acid killing that specifically sorts out cells expressing low levels of Na+/H+ antiport activity from a population of antiporter-deficient cells (AP-). LAP1 cells (TK- and AP-) were cotransformed with human genomic DNA and the thymidine kinase (TK) gene. TK+ transformants, first selected, were submitted to acid loading. The rare transformants that survived (frequency, 2-8 X 10(-7) expressed Na+/H+ antiport activity (AP+). We found that: transformation with mouse LAP1 DNA did not give rise to AP+ transformants; transformation of LAP1 cells with DNA from an altered Na+/H+ antiporter hamster variant led to AP+ transformants expressing the altered Na+/H+ antiporter of the DNA donor; human repeated sequences were present in all primary, secondary, and tertiary mouse AP+ transformants; six identical EcoRI human DNA fragments (55 kilobase pairs of the human genome) cosegregated with the Na+/H+ antiport activity in secondary and tertiary transformants. These results strongly suggest that we have stably expressed the structural gene for the human Na+/H+ antiporter in mouse cells.

Animals↗
PubMed1993

On the regulation of Na+/H+ and K+/H+ antiport in yeast mitochondria: evidence for the absence of an Na(+)-selective Na+/H+ antiporter.

Unlike mammalian mitochondria, yeast mitochondria swell spontaneously in both NaOAc and KOAc. This swelling reflects the activity of an electroneutral cation/H+ antiport pathway. Transport of neither salt is stimulated by depletion of endogenous divalent cations; however, it can be inhibited by addition of exogenous divalent cations (Mg2+ IC50 = 2.08 mM, Ca2+ IC50 = 0.82 mM). Transport of both Na+ and K+ can be completely inhibited by the amphiphilic amines propranolol (IC50 = 71 microM) and quinine (IC50 = 199 microM) with indistinguishable IC50 values. Dicyclohexylcarbodiimide inhibits with a second-order rate constant of 1.6 x 10(-4) (nmol DCCD/mg)-1 min-1 at 0 degrees C; however, with both Na+ and K+ inhibition reaches a maximum of about 46%. The remaining transport can still be inhibited by propranolol. Transport of both cations is sensitive to pH; yielding linear Hill plots and Dixon plots with a pIC50 value of 7.7 for both Na+ and K+. These properties are qualitatively the same as those of the non-selective K+/H+ antiporter of mammalian mitochondria. However, the remarkable similarity between the data obtained in Na+ and K+ media suggests that an antiporter akin to the Na(+)-selective Na+/H+ antiporter of mammalian mitochondria, which is inhibited by none of these agents, is absent in yeast. In an attempt to reveal the activity of a propranolol-insensitive Na(+)-selective antiporter, we compared the rates of Na+/H+ and K+/H+ antiport in the presence of sufficient propranolol to block the K+/H+ antiporter. Between pH 4.6 and 8.8 no difference could be detected. Consequently, we conclude that yeast mitochondria lack the typical Na(+)-selective Na+/H+ antiporter of mammalian mitochondria.

Antiporters↗
PubMed1988

Dual control of the intracellular pH in aortic smooth muscle cells by a cAMP-sensitive HCO3-/Cl- antiporter and a protein kinase C-sensitive Na+/H+ antiporter.

Two mechanisms are involved in the regulation of the intracellular pH (pHi) of aortic smooth muscle cells: the Na+/H+ antiporter and a Na+-independent HCO3-/Cl- antiporter. The Na+/H+ antiporter acts as a cell alkalinizing mechanism. It is activated by vasopressin and by phorbol esters when cells are incubated in the presence of bicarbonate but is not affected in the absence of bicarbonate. The HCO3-/Cl- antiporter acts as a cell acidifying mechanism. Agents such as forskolin, 8-Br-cAMP, and isoproterenol which raise intracellular cAMP levels inhibit the HCO3-/Cl- antiporter by shifting its pHi dependence in the alkaline direction. Thus, within the same cell type, different hormones control pHi variations by acting on different pHi regulating systems. An increase in pHi can be achieved either by a stimulation of a cell alkalinizing mechanism or by inhibition of a cell acidifying mechanism. A change of the activity of one pHi regulating mechanism modifies the responsiveness of the other to regulatory agents. Bicarbonate turns on the HCO3-/Cl- antiporter, decreases pHi and allows its regulation by protein kinase C through the Na+/H+ antiporter. Inhibition of the HCO3-/Cl- antiporter by cAMP increases the pHi and switches off the protein kinase C-mediated regulation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Role of chloride/bicarbonate antiport in the control of cytosolic pH. Cell-line differences in activity and regulation of antiport.

Sodium-linked and sodium-independent HCO3-/Cl- antiport was measured under different conditions in a number of cell lines. Transport of HCO3- was estimated from its effect on intracellular pH (pHi) measured with the fluorescent probe 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. The associated ion fluxes were estimated from the transport of 36Cl- and 22Na+. Na+-dependent and Na+-independent HCO3-/Cl- antiport were found in many, but not in all cell lines tested. The Na+-independent HCO3-/Cl- antiport was found to be highly pHi-dependent in a number of cell lines, whereas in others this was not the case. Some cell lines were found to have both Na+-dependent and Na+-independent HCO3-/Cl- antiport, whereas in others we could detect only one of these mechanisms. Na+/H+ antiport, which is quantitatively the most important H+-extruding mechanism, was found in all cell lines tested, but the activity varied strongly. Possible reasons for the qualitative and quantitative differences in antiport activity are discussed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1980

Cation/proton antiport systems in Escherichia coli. Properties of the potassium/proton antiporter.

The potassium/proton antiport system of Escherichia coli has been characterized by the effect of monovalent cations on the pH gradient formed by oxidation of lactate in everted membrane vesicles. Substrates of the system include K+, Na+, Li+, Rb+, and Tl+. The antiporter could also be assayed by uptake of 204Tl+ into everted vesicles. The antiporter exhibits a basic pH optimum and catalyzes electroneutral proton/cation exchange. Antiporter activity is trypsin-sensitive, but trypsin inactivation is prevented by prior formation of an electrochemical proton gradient. Two other proton/cation exchangers, the Na+/H+ and Ca2+/H+ antiporters, were unaffected by the trypsin treatment. Regulation of cytosolic pH by K+/H+ exchange is postulated, where proton return to the cytosol by the K+/H+ antiporter prevents alkalinization of the cytosol during proton extrusion associated with the formation of a protonmotive force or during growth at alkaline pH.

Biological Transport, Active↗
PubMed1999

Expression of functional Na+/H+ antiporters of Helicobacter pylori in antiporter-deficient Escherichia coli mutants.

An open reading frame with a sequence homologous to Escherichia coli Na+/H+ antiporter A (ENhaA) was found in the total genomic sequence of Helicobacter pylori, a pathogenic bacterium of gastric inflammation, and was named HNhaA. The primary sequences and the hydropathy profiles of ENhaA and HNhaA were very homologous except for one additional region found in HNhaA. This sequence has about 40 hydrophilic amino acid residues inserted at the position next to residue 235 of ENhaA which corresponds to residue 245 of HNhaA. HNhaA was expressed in E. coli mutants deficient in Na+/H+ antiporters and complemented the salt-sensitive phenotype of the mutants. Membrane vesicles prepared from these transformants of HNhaA using mutants deficient in the antiporters had the antiporter activities. Surprisingly, the antiporter activity in the transformant membranes was high at acidic and neutral pH, while ENhaA did not function at these pHs. A hydrophilic region around residue 235 in ENhaA and the additional hydrophilic region of about 40 residues in the same region found in HNhaA might be responsible for this difference in activity by acting as putative pH sensors.

Amino Acid Sequence↗
PubMed1990

Granulocyte-macrophage colony-stimulating factor can stimulate macrophage proliferation via persistent activation of Na+/H+ antiport. Evidence for two distinct roles for Na+/H+ antiport activation.

Macrophages respond to a variety of extracellular stimuli which can modulate the proliferation, development, activation and functional activity of these cells. The effects of two such agents, granulocytemacrophage colony-stimulating factor (GM-CSF, which stimulates proliferation) and platelet-activating factor (PAF, which stimulates chemotaxis and bactericidal activity), on cellular signal transduction mechanisms were compared. PAF can stimulate inositol lipid hydrolysis leading to Ca2+ mobilization. GM-CSF on the other hand has no effect on these events. Both agonists do, however, share an ability to activate an amiloride-sensitive Na+/H+ antiport and, furthermore, amiloride analogues are shown to inhibit the proliferative effects of GM-CSF on these cells. Long-term incubations with either PAF or GM-CSF demonstrate that it is only those cells pretreated with the latter which show a persistent activation of the antiport together with a sustained increase in intracellular pH. PAF-treated cells exhibit only a transitory increase in antiport activity, their intracellular pH levels returning to resting levels in spite of the continuous presence of the agonist in the medium. These effects of GM-CSF and PAF on Na+/H+ exchange are observed in both bicarbonate-free and bicarbonate-containing medium. These results lead us to suggest that the Na+/H+ antiport has a role in macrophage proliferation and in the regulation of intracellular pH during the oxidative burst stimulated by PAF and other agonists, and that differential mechanisms whereby this antiport is regulated exist in macrophages.

Animals↗
PubMed2001

nhaG Na(+)/H(+) antiporter gene of Bacillus subtilis ATCC9372, which is missing in the complete genome sequence of strain 168, and properties of the antiporter.

We cloned a gene which enabled Escherichia coli mutant host cells lacking all of the major Na(+)/H(+) antiporters to grow in the presence of 0.2 M NaCl from chromosomal DNA of Bacillus subtilis ATCC9372. An Na(+)/H(+) antiport activity was observed with membrane vesicles prepared from E. coli cells possessing the cloned gene, but not with vesicles from the host cells. Lithium ion was also a substrate for the antiporter. We sequenced the cloned DNA and found one open reading frame (designated nhaG) preceded by a promoter-like sequence and a Shine-Dalgarno sequence, and followed by a terminator-like sequence. The deduced amino acid sequence of NhaG suggested that it consisted of 524 residues and that the calculated molecular mass was 58.1 kDa. None of the bacterial Na(+)/H(+) antiporters so far reported, except NhaP of Pseudomonas aeruginosa and SynNhaP (NhaS1) of Synechocystis sp., showed significant sequence similarity with the NhaG. However, the NhaP, the SynNhaP, animal NHEs (Na(+)/H(+) exchangers), and some hypothetical Na(+)/H(+) antiporters of several organisms showed significant sequence similarities with the NhaG. Interestingly, the entire DNA region corresponding to the nhaG gene is missing in the reported complete genome sequence of B. subtilis strain 168. We detected a band that hybridized with the nhaG DNA in chromosomal DNA from B. subtilis ATCC9372 but not with that from strain 168. The missing DNA region (1,774 base pairs) is sandwiched by two identical sequences, TTTTCTT.

Animals↗
PubMed2001

Effect of the cationic detergent CTAB on the involvement of ADP/ATP antiporter and aspartate/glutamate antiporter in fatty acid-induced uncoupling of liver mitochondria.

The influence of the positively charged amphiphilic compound cetyltrimethyl ammonium bromide (CTAB) on palmitate- and laurate-induced uncoupling and on carboxyatractylate and glutamate recoupling effects in liver mitochondria have been studied. CTAB (40 microM) in the presence of 3 mM MgCl2 had little (if any) effect on the palmitic acid-stimulated respiration of mitochondria; the glutamate recoupling effect increased, and the carboxyatractylate recoupling effect decreased to the same degree with the combined effect (about 80%) remaining unchanged. Thus, CTAB decreases the ADP/ATP antiporter involvement and increases to the same extent the aspartate/glutamate antiporter involvement in the fatty acid-induced uncoupling. The carboxyatractylate and glutamate recoupling effects were less pH dependent in the presence of CTAB than in its absence. These data could be interpreted with the assumption that fatty acid anions are more accessible to the ADP/ATP antiporter and their neutral forms are more accessible to the aspartate/glutamate antiporter, and that CTAB changes the relative anion carrier involvement in the fatty acid-induced uncoupling as it forms neutral complexes with fatty acid anions.

Amino Acid Transport Systems, Acidic↗
PubMed1986

Cation/proton antiport systems in Escherichia coli. Solubilization and reconstitution of delta pH-driven sodium/proton and calcium/proton antiporters.

Uptake of 22Na+ and 45Ca2+ into everted membrane vesicles from Escherichia coli was measured with imposed transmembrane pH gradients, acid interior, as driving force. Vesicles loaded with 0.5 M KCl were diluted into 0.5 M choline chloride to create a potassium gradient. Addition of nigericin to produce K+/H+ exchange resulted in formation of a pH gradient. This imposed gradient was capable of driving 45Ca2+ accumulation. In another method vesicles loaded with 0.5 M NH4Cl were diluted into 0.5 M choline chloride, creating an ammonium diffusion potential. A gradient of H+ was produced by passive efflux of NH3. With an ammonium gradient as driving force, everted vesicles accumulated both 45Ca2+ and 22Na+. The data suggest that 22Na+ uptake was via the sodium/proton antiporter and 45Ca2+ via the calcium/proton antiporter. Uptake of both cations required alkaline pHout. A minimum pH gradient of 0.9 unit was needed for transport of either ion, suggesting gating of the antiporters. Octyl glucoside extracts of inner membrane were reconstituted with E. coli phospholipids in 0.5 M NH4Cl. NH4+-loaded proteoliposomes accumulated both 22Na+ and 45Ca2+, demonstrating that the sodium/proton and calcium/proton antiporters could be solubilized and reconstituted in a functional form.

Antiporters↗
PubMed1998

Evidence for chloramphenicol/H+ antiport in Cmr (MdfA) system of Escherichia coli and properties of the antiporter.

We detected chloramphenicol/H+ antiport activity in membrane vesicles of Escherichia coli and cloned a gene for the antiporter from chromosomal DNA of E. coli. Introduction of the gene into E. coli cells conferred resistance to chloramphenicol and ethidium. A slight increase in resistance to acridine orange was also observed. Elevated chloramphenicol efflux and ethidium efflux were observed in cells harboring a plasmid carrying the gene. Addition of chloramphenicol to the assay mixture reduced the efflux of ethidium. Elevated chloramphenicol/H+ antiport activity was observed in membrane vesicles prepared from cells harboring the plasmid. The pH optimum for the activity was 6.5. We sequenced the gene and deduced the amino acid sequence of its product. A sequence homology search revealed that it was same as that of Cmr (or MdfA). Thus, it became clear that Cmr (MdfA) is the chloramphenicol(and ethidium)/H+ antiporter.

Bacterial Proteins↗
PubMed1987

A dextran-bound amiloride derivative is a selective inhibitor of Na+/H+ antiport. Application for studying the role of the antiporter in cellular proliferation in human fibroblasts.

Inhibitors of Na+/H+ exchange from the amiloride series are known to accumulate within the cell and cause an inhibition of a variety of cellular functions. In order to render the amiloride molecule impermeable to cells, we have synthesized a potent amiloride analog, 5-N-(3-aminophenyl)amiloride (compound A35, Ki = 60 nM). The isothiocyanate derivative of A35 (A35-NCS) was coupled to soluble dextrans of 15-20 kDa that have been derivatized with diaminoalkane spacer groups. Dextran-bound amiloride derivatives showed good inhibition of Na+/H+ exchange in human foreskin fibroblasts and A431 cells. Among several spacer groups tested, dextran derivatized with ethylenediamine showed the highest inhibitory activity. The intrinsic inhibitory potency of this polymer increased with increasing degree of substitution with A35, approaching that of free A35 with substitution of approximately 3 mol of A35 per mole of dextran. Coupling to dextran largely diminished side effects of the amiloride derivative on cells such as the inhibition of protein synthesis. A35-dextran was an effective inhibitor of serum-induced reinitiation of DNA synthesis in human foreskin fibroblasts in a bicarbonate-free medium, pH 7.1, but had little effect when either the pH of the medium was more alkaline or when the medium contained a bicarbonate buffer. These findings suggest that the selective inhibition of Na+/H+ antiport by A35-dextran prevents the reinitiation of DNA synthesis when the external conditions are such that the antiporter activity is required for the establishment of a permissive intracellular pH. Polymer-bound amiloride analogs should be useful as selective inhibitors in studies of the physiological role of the Na+/H+ antiporter, as well as for affinity purification of the antiporter.

Amiloride↗
PubMed1995

Tetracycline/H+ antiport and Na+/H+ antiport catalyzed by the Bacillus subtilis TetA(L) transporter expressed in Escherichia coli.

The properties of TetA(L)-dependent tetracycline/proton and Na+/proton antiport were studied in energized everted vesicles of Escherichia coli transformed with a cloned tetA(L) gene (pJTA1) from Bacillus subtilis. Inhibition patterns by valinomycin and nigericin indicated that both antiports were electrogenic, in contrast to the tetracycline/proton antiport encoded by gram-negative plasmid tet genes. Tetracycline uptake in the everted system was dependent upon a divalent cation, with cobalt being the preferred one. The apparent Km for tetracycline was markedly increased at pH 8.5 versus pH 7.5, whereas the Vmax was unchanged. The much higher apparent Km for Na+ decreased at pH 8.5 relative to that at pH 7.5, as did the Vmax. Na+ did not affect tetracycline uptake, nor did Co2+ and/or tetracycline affect Na+ uptake; complex patterns of inhibition by amiloride and analogs thereof were observed.

Antiporters↗
PubMed1987

Stimulation by serum of the Na+/H+ antiporter in quiescent pig kidney epithelial (LLC-PK1) cells and role of the antiporter in the reinitiation of DNA synthesis.

LLC-PK1 cells can be brought into a classical quiescent state by depriving them of serum for 6 days. At this time, pulse-labeling with [3H]-thymidine shows that only 3% of the cells are synthesizing DNA, but the quiescent cells can be stimulated with serum to re-enter the cell cycle at a point early in G1. The rate of amiloride-sensitive 22Na+ uptake (as a measure of the Na+/H+ antiporter) is relatively low during quiescence; it rises 2- to 3-fold within 4 h after serum addition. This increase in antiporter activity appears to be required for the resumption of DNA synthesis in the absence of bicarbonate, because ethylisopropylamiloride (EIPA) blocks [3H]-thymidine incorporation when serum is added to cells in bicarbonate-free medium. In the presence of bicarbonate, however, EIPA has no effect on [3H]-thymidine incorporation, indicating that another (bicarbonate-dependent) transport system can substitute for the antiporter under these conditions.

Amiloride↗
PubMed1988

Reconstitution of the Na+/H+ antiporter: a new method for the determination of H+ efflux from Na+/H+ antiporter-reconstituted vesicles.

A Na+/H+ antiporter was solubilized with octyl-beta-D-glucopyranoside from rat renal brush border membrane and reconstituted in egg phosphatidylcholine liposomes. The activity of the antiporter was determined by a new method. Reconstitution was performed in a solution containing a pH-sensitive fluorescence dye, fluorescein isothiocyanate-dextran (FITC-dextran), at pH 5.5. The reconstituted vesicles, encapsulating FITC-dextran, were incubated at pH 8.0, and the intravesicular pH (pHi) was determined by monitoring fluorescence change of the dye. An increase in the fluorescence intensity which corresponded to an increase in pHi was observed. The rate of the increase was dependent on the concentration of the extravesicular Na+, and the efflux of H+ in the presence of 150 mM sodium gluconate was suppressed by the addition of amiloride. These results show that the Na+/H+ antiporter was functionally reconstituted and that its activity can be determined simply by the use of FITC-dextran.

Animals↗
PubMed1992

The putative Na+/H+ antiporter (NapA) of Enterococcus hirae is homologous to the putative K+/H+ antiporter (KefC) of Escherichia coli.

Two monovalent ion porters, the putative Na+/H+ antiporter (NapA) of Enterococcus hirae and the putative K+/H+ antiporter (KefC) of Escherichia coli, are similar in sequence throughout their hydrophobic domains. These two proteins, which comprise a novel family of transporters unrelated to the previously characterized Na+/H+ exchangers of E. coli (NhaA and NhaB) are proposed to function by essentially the same mechanism.

Amino Acid Sequence↗