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Hana Sychrová - One of the best experts on this subject based on the ideXlab platform.
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the salt tolerant yeast zygosaccharomyces rouxii possesses two plasma membrane na h Antiporters zrnha1p and zrsod2 22p playing different roles in cation homeostasis and cell physiology
Fungal Genetics and Biology, 2008Co-Authors: Lenka Pribylova, Klara Papouskova, Hana SychrováAbstract:Abstract Antiporters exporting Na+ and K+ in exchange for protons are conserved among yeast species. The only exception so far has been Zygosaccharomyces rouxii, an osmotolerant species closely related to Saccharomyces cerevisiae. Z. rouxii was described as possessing one plasma-membrane Antiporter transporting only Na+ (ZrSod2–22p in the CBS 732T type strain). We report the characterization of a second gene, ZrNHA1, encoding a new K+(Na+)/H+-Antiporter capable of both K+ and Na+ export. Synteny analyses suggested that ZrSOD2–22 originated by single duplication of the ZrNHA1 gene. Substrate specificities and transport properties of ZrNha1p and ZrSod2–22p were compared upon heterologous expression in S. cerevisiae, and then directly in Z. rouxii. Deletion mutants and phenotype analyses revealed that ZrSod2–22 Antiporter is important for Na+ detoxification, probably together with ZrEna1 ATPase; ZrNha1p is indispensable to maintain potassium homeostasis and ZrEna1p is not, in contrast to the situation in S. cerevisiae, involved in this function.
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Schizosaccharomyces pombe possesses two plasma membrane alkali metal cation/H Antiporters differing in their substrate specificity.
Fems Yeast Research, 2007Co-Authors: Klara Papouskova, Hana SychrováAbstract:: The Schizosaccharomyces pombe plasma membrane Na(+)/H(+) Antiporter, SpSod2p, has been shown to belong to the subfamily of yeast Na(+)/H(+) Antiporters that only recognize Na(+) and Li(+) as substrates. Nevertheless, most of the studied plasma membrane alkali metal cation/H(+) Antiporters from other yeasts have broader substrate specificities, exporting K(+) and Rb(+) as well. Such Antiporters probably play two roles in the physiology of cells: the elimination of surplus toxic cations, and the regulation of stable intracellular K(+) content, pH and cell volume. The systematic sequencing of the Sch. pombe genome revealed the presence of an as-yet uncharacterized homolog of the Spsod2 gene (designated Spsod22). Spsod22 and Spsod2 were expressed in Saccharomyces cerevisiae cells lacking their own alkali metal cation efflux systems, and the transport properties of both Sch. pombe Antiporters were compared to those of the Sac. cerevisiae Nha1 Antiporter expressed under the same conditions. Here we show that SpSod22p has broad substrate specificity upon heterologous expression in Sac. cerevisiae cells and contributes to cell tolerance to high external levels of K(+). Thus, the Sch. pombe genome encodes two plasma membrane alkali metal cation/H(+) Antiporters that play different roles in the physiology of the yeast.
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yarrowia lipolytica possesses two plasma membrane alkali metal cation h Antiporters with different functions in cell physiology
FEBS Letters, 2006Co-Authors: Klara Papouskova, Hana SychrováAbstract:The family of Nha Antiporters mediating the efflux of alkali metal cations in exchange for protons across the plasma membrane is conserved in all yeast species. Yarrowia lipolytica is a dimorphic yeast, phylogenetically very distant from the model yeast Saccharomyces cerevisiae. A search in its sequenced genome revealed two genes (designated as YlNHA1 and YlNHA2) with homology to the S. cerevisiae NHA1 gene, which encodes a plasma membrane alkali metal cation/H+ Antiporter. Upon heterologous expression of both YlNHA genes in S. cerevisiae, we showed that Y. lipolytica Antiporters differ not only in length and sequence, but also in their affinity for individual substrates. While the YlNha1 protein mainly increased cell tolerance to potassium, YlNha2p displayed a remarkable transport capacity for sodium. Thus, Y. lipolytica is the first example of a yeast species with two plasma membrane alkali metal cation/H+ Antiporters differing in their putative functions in cell physiology; cell detoxification vs. the maintenance of stable intracellular pH, potassium content and cell volume.
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the debaryomyces hansenii nha1 gene encodes a plasma membrane alkali metal cation Antiporter with broad substrate specificity
Gene, 2006Co-Authors: Klara Velkova, Hana SychrováAbstract:Abstract Debaryomyces hansenii is a yeast species often found in salty environments. Its genome sequence is known completely, but the mechanisms behind its halotolerance are poorly understood. In the D. hansenii genome, there is a gene strongly homologous to the Saccharomyces cerevisiae NHA1 gene (encoding a plasma membrane Na+/H+ Antiporter). We isolated this DhNHA1 gene from two D. hansenii strains (CBS 767 and CBS 1793) differing in their osmotolerance. Both DhNHA1 alleles were heterologously expressed in a S. cerevisiae strain lacking its own systems for the efflux of alkali metal cations (BW31a, ena1–4Δ nha1Δ). D. hansenii Na+/H+ Antiporters were localized in the plasma membrane of BW31a cells, their presence increased BW31a tolerance to sodium, potassium, lithium and also rubidium. Measurements of Na+ and K+ efflux from S. cerevisiae cells expressing DhNHA1 alleles show that the D. hansenii Antiporters efficiently transported both cations out of cells. The sodium and potassium transport activity of Nha1 Antiporters from both D. hansenii strains was almost identical, indicating that plasma membrane Antiporter activity is not one of the factors determining the different levels of halotolerance in the two strains.
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Yarrowia lipolytica possesses two plasma membrane alkali metal cation/H+ Antiporters with different functions in cell physiology.
FEBS Letters, 2006Co-Authors: Klara Papouskova, Hana SychrováAbstract:Abstract The family of Nha Antiporters mediating the efflux of alkali metal cations in exchange for protons across the plasma membrane is conserved in all yeast species. Yarrowia lipolytica is a dimorphic yeast, phylogenetically very distant from the model yeast Saccharomyces cerevisiae. A search in its sequenced genome revealed two genes (designated as YlNHA1 and YlNHA2) with homology to the S. cerevisiae NHA1 gene, which encodes a plasma membrane alkali metal cation/H+ Antiporter. Upon heterologous expression of both YlNHA genes in S. cerevisiae, we showed that Y. lipolytica Antiporters differ not only in length and sequence, but also in their affinity for individual substrates. While the YlNha1 protein mainly increased cell tolerance to potassium, YlNha2p displayed a remarkable transport capacity for sodium. Thus, Y. lipolytica is the first example of a yeast species with two plasma membrane alkali metal cation/H+ Antiporters differing in their putative functions in cell physiology; cell detoxification vs. the maintenance of stable intracellular pH, potassium content and cell volume.
Etana Padan - One of the best experts on this subject based on the ideXlab platform.
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sodium proton na h Antiporters properties and roles in health and disease
Metal ions in life sciences, 2016Co-Authors: Etana Padan, Meytal LandauAbstract:The transmembranal Na+/H+ Antiporters transport sodium (or several other monovalent cations) in exchange for H+ across lipid bilayers in all kingdoms of life. They are critical in pH homeostasis of the cytoplasm and/or organelles. A particularly notable example is the SLC9 gene family, which encodes Na+/H+ exchangers (NHEs) in many species from prokaryotes to eukaryotes. In humans, these proteins are associated with the pathophysiology of various diseases. Yet, the most extensively studied Na+/H+ Antiporter is Ec-NhaA, the main Na+/H+ Antiporter of Escherichia coli.
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revealing the ligand binding site of nhaa na h Antiporter and its ph dependence
Journal of Biological Chemistry, 2012Co-Authors: Michal Maes, Abraham Rimon, Lena Kozachkovmagrisso, Assaf Friedler, Etana PadanAbstract:pH and Na(+) homeostasis in all cells requires Na(+)/H(+) Antiporters. In most cases, their activity is tightly pH-regulated. NhaA, the main Antiporter of Escherichia coli, has homologues in all biological kingdoms. The crystal structure of NhaA provided insights into the mechanism of action and pH regulation of an Antiporter. However, the active site of NhaA remained elusive because neither Na(+) nor Li(+), the NhaA ligands, were observed in the structure. Using isothermal titration calorimetry, we show that purified NhaA binds Li(+) in detergent micelles. This interaction is driven by an increase in enthalpy (ΔH of -8000 ± 300 cal/mol and ΔS of -15.2 cal/mol/degree at 283 K), involves a single binding site per NhaA molecule, and is highly specific and drastically dependent on pH; Li(+) binding was observed only at pH 8.5. Combining mutational analysis with the isothermal titration calorimetry measurements revealed that Asp-163, Asp-164, Thr-132, and Asp-133 form the Li(+) binding site, whereas Lys-300 plays an important role in pH regulation of the Antiporter.
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The enlightening encounter between structure and function in the NhaA Na+–H+ Antiporter
Trends in biochemical sciences, 2008Co-Authors: Etana PadanAbstract:Na + –H + Antiporters are integral membrane proteins that exchange Na + for H + across the cytoplasmic membrane and many intracellular membranes. They are essential for Na + , pH, and volume homeostasis, which are processes crucial for cell viability. Accordingly, Antiporters are important drug targets in humans and underlie salt resistance in plants. Many Na + –H + Antiporters are tightly regulated by pH. Escherichia coli NhaA, a prototype pH-regulated Antiporter, exchanges 2H + for 1Na + (or Li + ). The NhaA crystal structure has provided insight into the pH-regulated mechanism of Antiporter action and revealed transmembrane segments, which are interrupted by extended mid-membrane chains that have since been found with variations in other ion-transport proteins. This novel structural fold creates a delicately balanced electrostatic environment in the middle of the membrane, which might be essential for ion binding and translocation.
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structure of a na h Antiporter and insights into mechanism of action and regulation by ph
Nature, 2005Co-Authors: Carola Hunte, Etana Padan, Miro Venturi, Abraham Rimon, Emanuela Screpanti, Hartmut MichelAbstract:The control by Na+/H+ Antiporters of sodium/proton concentration and cell volume is crucial for the viability of all cells. Adaptation to high salinity and/or extreme pH in plants and bacteria or in human heart muscles requires the action of Na+/H+ Antiporters. Their activity is tightly controlled by pH. Here we present the crystal structure of pH-downregulated NhaA, the main Antiporter of Escherichia coli and many enterobacteria. A negatively charged ion funnel opens to the cytoplasm and ends in the middle of the membrane at the putative ion-binding site. There, a unique assembly of two pairs of short helices connected by crossed, extended chains creates a balanced electrostatic environment. We propose that the binding of charged substrates causes an electric imbalance, inducing movements, that permit a rapid alternating-access mechanism. This ion-exchange machinery is regulated by a conformational change elicited by a pH signal perceived at the entry to the cytoplasmic funnel.
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roles of nhaa nhab and nhad na h Antiporters in survival of vibrio cholerae in a saline environment
Journal of Bacteriology, 2003Co-Authors: Katia Herz, Etana Padan, Sophie Vimont, Patrick BercheAbstract:Vibrio cholerae, the causative agent of cholera, is a normal inhabitant of aquatic environments, where it survives in a wide range of conditions of pH and salinity. In this work, we investigated the role of three Na+/H+ Antiporters on the survival of V. cholerae in a saline environment. We have previously cloned the Vc-nhaA gene encoding the V. cholerae homolog of Escherichia coli. Here we identified two additional Antiporter genes, designated Vc-nhaB and Vc-nhaD, encoding two putative proteins of 530 and 477 residues, respectively, highly homologous to the respective Antiporters of Vibrio species and E. coli. We showed that both Vc-NhaA and Vc-NhaB confer Na+ resistance and that Vc-NhaA displays an antiport activity in E. coli, which is similar in magnitude, kinetic parameters, and pH regulation to that of E. coli NhaA. To determine the roles of the Na+/H+ Antiporters in V. cholerae, we constructed nhaA, nhaB, and nhaD mutants (single, double, and triple mutants). In contrast to E. coli, the inactivation of the three putative Antiporter genes (Vc-nhaABD) in V. cholerae did not alter the bacterial exponential growth in the presence of high Na+ concentrations and had only a slight effect in the stationary phase. In contrast, a pronounced and similar Li+-sensitive phenotype was found with all mutants lacking Vc-nhaA during the exponential phase of growth and also with the triple mutant in the stationary phase of growth. By using 2-n-nonyl-4-hydroxyquinoline N-oxide, a specific inhibitor of the electron-transport-linked Na+ pump NADH-quinone oxidoreductase (NQR), we determined that in the absence of NQR activity, the Vc-NhaA Na+/H+ Antiporter activity becomes essential for the resistance of V. cholerae to Na+ at alkaline pH. Since the ion pump NQR is Na+ specific, we suggest that its activity masks the Na+/H+ but not the Li+/H+ Antiporter activities. Our results indicate that the Na+ resistance of the human pathogen V. cholerae requires a complex molecular system involving multiple Antiporters and the NQR pump.
Tomofusa Tsuchiya - One of the best experts on this subject based on the ideXlab platform.
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Expression of functional Na+/H+ Antiporters of Helicobacter pylori in Antiporter-deficient Echerichia coli mutants
FEBS letters, 1999Co-Authors: Hiroki Inoue, Tomofusa Tsuchiya, Tatsuya Sakurai, Satoshi Ujike, Hiroshi Murakami, Hiroshi KanazawaAbstract: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.
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Expression of functional Na á /H á Antiporters of Helicobacter pylori in Antiporter-de¢cient Echerichia coli mutants
1999Co-Authors: Hiroki Inoue, Tomofusa Tsuchiya, Tatsuya Sakurai, Satoshi Ujike, Hiroshi Murakami, Hiroshi KanazawaAbstract: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. z 1999 Federation of European Biochemical Societies. essential for understanding the relationship between structure and function of ENhaA. Since the activity of ENhaA depends on the pH outside membrane vesicles, surveying the characteristics of Antiporters in other bacteria living in pH environments diierent from that of E. coli could provide some insights into the functioning of ENhaA including mechanisms of the hypothetical pH sensor. From this point of view, we surveyed a homologous sequence of NhaA in Helicobacter pylori (13) which is known as a pathogenic bacterium of gastric in£ammation that lives under very acidic conditions. We found one open reading frame that has a sequence homologous to that of ENhaA (HNhaA). We have subcloned the sequence (HNhaA) into an expression vector of E. coli and introduced it into E. coli mutants de¢- cient in the Antiporters. HNhaA complemented the defective phenotype of an E. coli mutant whose growth is inhibited by LiCl and high concentrations of NaCl. HNhaA in E. coli membrane vesicles, unlike ENhaA, exhibited a high activity in the pH range from 6.0 to 8.5.
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A Putative Multisubunit Na+/H+ Antiporter from Staphylococcus aureus
Journal of bacteriology, 1998Co-Authors: Toshiaki Hiramatsu, Teruo Kuroda, Tohru Mizushima, Kazuyo Kodama, Tomofusa TsuchiyaAbstract:We cloned several genes encoding an Na+/H+ Antiporter of Staphylococcus aureus from chromosomal DNA by using an Escherichia coli mutant, lacking all of the major Na+/H+ Antiporters, as the host. E. coli cells harboring plasmids for the cloned genes were able to grow in medium containing 0.2 M NaCl (or 10 mM LiCl). Host cells without the plasmids were unable to grow under the same conditions. Na+/H+ antiport activity was detected in membrane vesicles prepared from transformants. We determined the nucleotide sequence of the cloned 7-kbp region. We found that seven open reading frames (ORFs) were necessary for Antiporter function. A promoter-like sequence was found in the upstream region from the first ORF. One inverted repeat followed by a T-cluster, which may function as a terminator, was found in the downstream region from the seventh ORF. Neither terminator-like nor promoter-like sequences were found between the ORFs. Thus, it seems that the seven ORFs comprise an operon and that the Na+/H+ Antiporter consists of seven kinds of subunits, suggesting that this is a novel type of multisubunit Na+/H+ Antiporter. Hydropathy analysis of the deduced amino acid sequences of the seven ORFs suggested that all of the proteins are hydrophobic. As a result of a homology search, we found that components of the respiratory chain showed sequence similarity with putative subunits of the Na+/H+ Antiporter. We observed a large Na+ extrusion activity, driven by respiration in E. coli cells harboring the plasmid carrying the genes. The Na+ extrusion was sensitive to an H+ conductor, supporting the idea that the system is not a respiratory Na+ pump but an Na+/H+ Antiporter. Introduction of the plasmid into E. coli mutant cells, which were unable to grow under alkaline conditions, enabled the cells to grow under such conditions.
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A NEW NA+/H+ Antiporter, NHAD, OF VIBRIO PARAHAEMOLYTICUS
Biochimica et biophysica acta, 1998Co-Authors: Kaori Nozaki, Teruo Kuroda, Tohru Mizushima, Tomofusa TsuchiyaAbstract:A gene encoding an Na+/H+ Antiporter was cloned from chromosomal DNA of Vibrio parahaemolyticus, a slightly halophilic bacterium, and expressed in Escherichia coli cells. The gene enabled mutant E. coli cells, which were unable to grow in the presence of 10 mM LiCl (or 0.2 M NaCl) because of the lack of major Na+(Li+)/H+ Antiporters, to grow under such conditions. We detected Na+/H+ antiport activity due to the gene in membrane vesicles prepared from E. coli cells that harbored the plasmid carrying the gene. Li+ was also a substrate for this Antiporter. Activity of this Antiporter was pH-dependent with highest activity at pH 8.5 to 9 and no activity at 7.0 to 7.5. Restriction mapping and a Southern blot analysis revealed that the cloned gene was different from the nhaA and the nhaB of V. parahaemolyticus. We designated the gene nhaD. The gene was sequenced, and the amino acid sequence of the NhaD protein was deduced. The NhaD is a unique Na+/H+ Antiporter with respect to the primary structure compared with known Na+/H+ Antiporters.
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pH-dependent growth retardation of Escherichia coli caused by overproduction of Na+/H+ Antiporter.
Biological & pharmaceutical bulletin, 1998Co-Authors: Hiroki Inoue, Tomofusa Tsuchiya, Takato Noumi, Tetsuya Shimomura, Nobuya Takimoto, Hiroshi KanazawaAbstract:The overproduction of Na+/H+ Antiporter NhaA in Escherichia coli caused growth retardation. Quantities and the activity of the Antiporter were studied for their causative roles in terms of this retardation. We constructed a series of nhaA-expression plasmids differing in their transcriptional and translational efficiencies. Low-level nhaA expression complemented the defect of an nhaA mutant and allowed the mutant to survive on a high-NaCl or high-LiCl medium. However, when the production of NhaA was strongly induced by the combination of a stronger promoter, an efficient translational initiation signal and a high copy number plasmid, the growth of the cells carrying the plasmid was severely retarded. This growth retardation correlated well with the amount of NhaA protein produced from the plasmids. Surprisingly, the growth retardation caused by overproduction of NhaA was enhanced more extensively at an alkaline pH than at a neutral pH, in which the Antiporter activity was stimulated. However, these retardations were also observed for mutant NhaA Antiporters without the activity. These results indicated that the growth retardation was due to an overproduction of the Antiporter rather than its increased Antiporter activity, and also affected by a pH-dependent change in NhaA, possibly its structural change.
Pavel Dibrov - One of the best experts on this subject based on the ideXlab platform.
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insights into the biochemistry of the ubiquitous nhap family of cation h Antiporters 1
Biochemistry and Cell Biology, 2011Co-Authors: Craig T. Resch, Judith L. Winogrodzki, Pavel DibrovAbstract:Na + /H + Antiporters are integral membrane proteins that exchange Na + for H + across the cytoplasmic or organel- lar membranes of virtually all living cells. They are essential for control of cellular pH, volume homeostasis, and regula- tion of Na + levels. Na + /H + Antiporters have become increasingly characterized and are now becoming important drug targets. The recently identified NhaP family of Na + /H + Antiporters, from the CPA1 superfamily, contains proteins with a surprisingly broad collective range of transported cations, exchanging protons for alkali cations such as Na + ,L i + ,K + ,o r Rb + as well as for Ca 2+ and, possibly, NH4 + . Questions about ion selectivity and the physiological impact of each particu- lar NhaP Antiporter are far from trivial. For example, Vc-NhaP2 from Vibrio cholerae has recently been shown to function in vivo as a specific K + /H + Antiporter while retaining the ability to exchange H + for Na + and bind (but not exchange with H + )L i + in a competitive manner. These and other findings reviewed in this communication make Antiporters of the NhaP type attractive systems to study intimate molecular mechanisms of cation exchange. In an evolutionary perspective, the NhaP family seems to be a phylogenetic entity undergoing active divergent evolution. In this minireview, to rationalize pe- culiarities of the cation specificity in the NhaP family, the ''size-exclusion principle'' and the idea of ''ligand shading'' are discussed.
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Insights into the biochemistry of the ubiquitous NhaP family of cation/H + Antiporters 1
Biochemistry and cell biology = Biochimie et biologie cellulaire, 2011Co-Authors: Craig T. Resch, Judith L. Winogrodzki, Pavel DibrovAbstract:Na + /H + Antiporters are integral membrane proteins that exchange Na + for H + across the cytoplasmic or organel- lar membranes of virtually all living cells. They are essential for control of cellular pH, volume homeostasis, and regula- tion of Na + levels. Na + /H + Antiporters have become increasingly characterized and are now becoming important drug targets. The recently identified NhaP family of Na + /H + Antiporters, from the CPA1 superfamily, contains proteins with a surprisingly broad collective range of transported cations, exchanging protons for alkali cations such as Na + ,L i + ,K + ,o r Rb + as well as for Ca 2+ and, possibly, NH4 + . Questions about ion selectivity and the physiological impact of each particu- lar NhaP Antiporter are far from trivial. For example, Vc-NhaP2 from Vibrio cholerae has recently been shown to function in vivo as a specific K + /H + Antiporter while retaining the ability to exchange H + for Na + and bind (but not exchange with H + )L i + in a competitive manner. These and other findings reviewed in this communication make Antiporters of the NhaP type attractive systems to study intimate molecular mechanisms of cation exchange. In an evolutionary perspective, the NhaP family seems to be a phylogenetic entity undergoing active divergent evolution. In this minireview, to rationalize pe- culiarities of the cation specificity in the NhaP family, the ''size-exclusion principle'' and the idea of ''ligand shading'' are discussed.
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the putative na h Antiporter of vibrio cholerae vc nhap2 mediates the specific k h exchange in vivo
Biochemistry, 2010Co-Authors: Craig T. Resch, Judith L. Winogrodzki, Pavel Dibrov, Curtis T Patterson, Erin J Lind, Matthew J Quinn, Claudia C HaseAbstract:Potassium is the major monovalent cation of the bacterial cytoplasm. It regulates internal pH, activates many intracellular enzymes and functions as an important osmotic solute (1). However, excessive amounts of internal K+ are detrimental (2–4). Therefore, bacteria tightly regulate their cytoplasmic K+ through the activity of a number of different transport systems (reviewed in (1)). Kdp, Trk and Kup systems import K+ either at the expense of ATP hydrolysis (TrkA and Kdp) or symporting it with a proton (Kup) (5–8). In addition, tetracycline Antiporters TetL in Bacillus subtilis and TetK in Staphylococcus aureus that are able to exchange monovalent cations, may contribute to the net K+ uptake (9–11). Export of K+ can be mediated by (a) the glutathione adduct-activated “emergency” KefB/KefC systems of Gram-negative organisms (12); (b) mechanosensitive channels under severe hypoosmotic stress (13–15), although they are thought to play only a minor role in overall K+ homeostasis (1); and (c) the MdfA multidrug resistance transporter, which at external pH >9.0 import protons in exchange for extracellular Na+ or K+ (16). All the above potassium-expelling systems seem to be mobilized only in specific stressful situations. Paradoxically, the identity of system(s) responsible for routine energy-dependent K+ extrusion remains poorly understood. Almost fifty years ago, Peter Mitchell postulated the existence of “housekeeping” K+/H+ and Na+/H+ Antiporters, that can directly use the proton motive force to prevent the dangerous over-accumulation of alkali cations (17). Typically, growing bacteria employ a variety of primary proton pumps to maintain a high transmembrane electrical potential difference, ΔΨ (negative inside) over a wide range of external pH. As a result, K+ (or any other monovalent cation), if allowed to equilibrate with the ΔΨ, would accumulate inside the cell at poisonous concentrations. At −120 mV of ΔΨ and a moderate external K+ concentration of 30 mM, at equilibrium the cell would accumulate as much as 3 M K+, a concentration that clearly is beyond the physiological limit. A K+/H+ Antiporter would allow H+ expelled by the primary pumps to return into the cytoplasm in exchange for internal K+, thus solving the problem. Although several families of bacterial Na+/H+ Antiporters have been identified and studied in great detail (18–22), identification of specific K+/H+ Antiporters in bacteria remains elusive. K+/H+ antiport activity as such has been demonstrated in everted membrane vesicles from E. coli a long time ago (23). Some Na+/H+ Antiporters, exemplified by well-studied Ec-NhaA and Ec-NhaB (22), are highly discriminative against K+, while others exhibit more or less pronounced K+/H+ exchange as a concomitant activity, such as the multi-subunit Vc-Mrp in Vibrio cholerae (24), or the alkali-activated Aa-NhaP from Alkalimonas amylolytica that transports Na+, K+ and possibly NH4+, but not Li+ (25). Recently, Radchenko and co-authors reported that Vp-NhaP2 from V. parahaemolyticus might be a K+-specific Antiporter (4). If confirmed, this would set a valuable precedent, because in spite of the widely recognized importance of K+/H+ Antiporters for bacterial ion and pH homeostasis (1), no transporter exclusively specific for K+ has been identified thus far. The authors assayed inside-out vesicles obtained from Antiporter-deficient E. coli overexpressing the cloned Vp-NhaP2. The Antiporter displayed a rather modest activity with K+ even at its pH optimum of 9.0; in the absence of K+, Na+ seemed to be a substrate as well, albeit poorer than K+ (see Fig. 5B in (4)). Unfortunately, the authors did not examine the effect of Na+ concentration on the Na+/H+ antiport activity. Therefore, definitive conclusions about the specificity of Vp-NhaP2 were hard to make at the moment. Also, one more pressing question remained: would the chromosomal deletion of nhaP2 gene produce a potassium-sensitive phenotype in its native host, V. parahaemolyticus? Inspired by the work of Radchenko and colleagues, we undertook a search for other possible Antiporters exclusively transporting K+. In the course of this search we cloned, functionally expressed and examined a homologue of Vp-NhaP2 from Vibrio cholerae O395, Vc-NhaP2, encoded by the open reading frame VC2703. We also engineered and characterized the Vp-NhaP2 chromosomal deletion mutant of V. cholerae. Data presented in this article define Vc-NhaP2 as an electroneutral K+/H+ Antiporter, which in vitro is able to catalyze K+/H+, Rb+/H+, Na+/H+ and, possibly, Li+/K+ (but not Li+/H+) exchange, but in situ operates as a Mitchellian K+/H+ Antiporter, protecting V. cholerae cells growing at pH 6.0 from high concentrations of K+. The peculiar behavior of Vc-NhaP2 in relation to the general problem of search for specific K+/H+ Antiporters in bacteria is discussed.
Craig T. Resch - One of the best experts on this subject based on the ideXlab platform.
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insights into the biochemistry of the ubiquitous nhap family of cation h Antiporters 1
Biochemistry and Cell Biology, 2011Co-Authors: Craig T. Resch, Judith L. Winogrodzki, Pavel DibrovAbstract:Na + /H + Antiporters are integral membrane proteins that exchange Na + for H + across the cytoplasmic or organel- lar membranes of virtually all living cells. They are essential for control of cellular pH, volume homeostasis, and regula- tion of Na + levels. Na + /H + Antiporters have become increasingly characterized and are now becoming important drug targets. The recently identified NhaP family of Na + /H + Antiporters, from the CPA1 superfamily, contains proteins with a surprisingly broad collective range of transported cations, exchanging protons for alkali cations such as Na + ,L i + ,K + ,o r Rb + as well as for Ca 2+ and, possibly, NH4 + . Questions about ion selectivity and the physiological impact of each particu- lar NhaP Antiporter are far from trivial. For example, Vc-NhaP2 from Vibrio cholerae has recently been shown to function in vivo as a specific K + /H + Antiporter while retaining the ability to exchange H + for Na + and bind (but not exchange with H + )L i + in a competitive manner. These and other findings reviewed in this communication make Antiporters of the NhaP type attractive systems to study intimate molecular mechanisms of cation exchange. In an evolutionary perspective, the NhaP family seems to be a phylogenetic entity undergoing active divergent evolution. In this minireview, to rationalize pe- culiarities of the cation specificity in the NhaP family, the ''size-exclusion principle'' and the idea of ''ligand shading'' are discussed.
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Insights into the biochemistry of the ubiquitous NhaP family of cation/H + Antiporters 1
Biochemistry and cell biology = Biochimie et biologie cellulaire, 2011Co-Authors: Craig T. Resch, Judith L. Winogrodzki, Pavel DibrovAbstract:Na + /H + Antiporters are integral membrane proteins that exchange Na + for H + across the cytoplasmic or organel- lar membranes of virtually all living cells. They are essential for control of cellular pH, volume homeostasis, and regula- tion of Na + levels. Na + /H + Antiporters have become increasingly characterized and are now becoming important drug targets. The recently identified NhaP family of Na + /H + Antiporters, from the CPA1 superfamily, contains proteins with a surprisingly broad collective range of transported cations, exchanging protons for alkali cations such as Na + ,L i + ,K + ,o r Rb + as well as for Ca 2+ and, possibly, NH4 + . Questions about ion selectivity and the physiological impact of each particu- lar NhaP Antiporter are far from trivial. For example, Vc-NhaP2 from Vibrio cholerae has recently been shown to function in vivo as a specific K + /H + Antiporter while retaining the ability to exchange H + for Na + and bind (but not exchange with H + )L i + in a competitive manner. These and other findings reviewed in this communication make Antiporters of the NhaP type attractive systems to study intimate molecular mechanisms of cation exchange. In an evolutionary perspective, the NhaP family seems to be a phylogenetic entity undergoing active divergent evolution. In this minireview, to rationalize pe- culiarities of the cation specificity in the NhaP family, the ''size-exclusion principle'' and the idea of ''ligand shading'' are discussed.
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the putative na h Antiporter of vibrio cholerae vc nhap2 mediates the specific k h exchange in vivo
Biochemistry, 2010Co-Authors: Craig T. Resch, Judith L. Winogrodzki, Pavel Dibrov, Curtis T Patterson, Erin J Lind, Matthew J Quinn, Claudia C HaseAbstract:Potassium is the major monovalent cation of the bacterial cytoplasm. It regulates internal pH, activates many intracellular enzymes and functions as an important osmotic solute (1). However, excessive amounts of internal K+ are detrimental (2–4). Therefore, bacteria tightly regulate their cytoplasmic K+ through the activity of a number of different transport systems (reviewed in (1)). Kdp, Trk and Kup systems import K+ either at the expense of ATP hydrolysis (TrkA and Kdp) or symporting it with a proton (Kup) (5–8). In addition, tetracycline Antiporters TetL in Bacillus subtilis and TetK in Staphylococcus aureus that are able to exchange monovalent cations, may contribute to the net K+ uptake (9–11). Export of K+ can be mediated by (a) the glutathione adduct-activated “emergency” KefB/KefC systems of Gram-negative organisms (12); (b) mechanosensitive channels under severe hypoosmotic stress (13–15), although they are thought to play only a minor role in overall K+ homeostasis (1); and (c) the MdfA multidrug resistance transporter, which at external pH >9.0 import protons in exchange for extracellular Na+ or K+ (16). All the above potassium-expelling systems seem to be mobilized only in specific stressful situations. Paradoxically, the identity of system(s) responsible for routine energy-dependent K+ extrusion remains poorly understood. Almost fifty years ago, Peter Mitchell postulated the existence of “housekeeping” K+/H+ and Na+/H+ Antiporters, that can directly use the proton motive force to prevent the dangerous over-accumulation of alkali cations (17). Typically, growing bacteria employ a variety of primary proton pumps to maintain a high transmembrane electrical potential difference, ΔΨ (negative inside) over a wide range of external pH. As a result, K+ (or any other monovalent cation), if allowed to equilibrate with the ΔΨ, would accumulate inside the cell at poisonous concentrations. At −120 mV of ΔΨ and a moderate external K+ concentration of 30 mM, at equilibrium the cell would accumulate as much as 3 M K+, a concentration that clearly is beyond the physiological limit. A K+/H+ Antiporter would allow H+ expelled by the primary pumps to return into the cytoplasm in exchange for internal K+, thus solving the problem. Although several families of bacterial Na+/H+ Antiporters have been identified and studied in great detail (18–22), identification of specific K+/H+ Antiporters in bacteria remains elusive. K+/H+ antiport activity as such has been demonstrated in everted membrane vesicles from E. coli a long time ago (23). Some Na+/H+ Antiporters, exemplified by well-studied Ec-NhaA and Ec-NhaB (22), are highly discriminative against K+, while others exhibit more or less pronounced K+/H+ exchange as a concomitant activity, such as the multi-subunit Vc-Mrp in Vibrio cholerae (24), or the alkali-activated Aa-NhaP from Alkalimonas amylolytica that transports Na+, K+ and possibly NH4+, but not Li+ (25). Recently, Radchenko and co-authors reported that Vp-NhaP2 from V. parahaemolyticus might be a K+-specific Antiporter (4). If confirmed, this would set a valuable precedent, because in spite of the widely recognized importance of K+/H+ Antiporters for bacterial ion and pH homeostasis (1), no transporter exclusively specific for K+ has been identified thus far. The authors assayed inside-out vesicles obtained from Antiporter-deficient E. coli overexpressing the cloned Vp-NhaP2. The Antiporter displayed a rather modest activity with K+ even at its pH optimum of 9.0; in the absence of K+, Na+ seemed to be a substrate as well, albeit poorer than K+ (see Fig. 5B in (4)). Unfortunately, the authors did not examine the effect of Na+ concentration on the Na+/H+ antiport activity. Therefore, definitive conclusions about the specificity of Vp-NhaP2 were hard to make at the moment. Also, one more pressing question remained: would the chromosomal deletion of nhaP2 gene produce a potassium-sensitive phenotype in its native host, V. parahaemolyticus? Inspired by the work of Radchenko and colleagues, we undertook a search for other possible Antiporters exclusively transporting K+. In the course of this search we cloned, functionally expressed and examined a homologue of Vp-NhaP2 from Vibrio cholerae O395, Vc-NhaP2, encoded by the open reading frame VC2703. We also engineered and characterized the Vp-NhaP2 chromosomal deletion mutant of V. cholerae. Data presented in this article define Vc-NhaP2 as an electroneutral K+/H+ Antiporter, which in vitro is able to catalyze K+/H+, Rb+/H+, Na+/H+ and, possibly, Li+/K+ (but not Li+/H+) exchange, but in situ operates as a Mitchellian K+/H+ Antiporter, protecting V. cholerae cells growing at pH 6.0 from high concentrations of K+. The peculiar behavior of Vc-NhaP2 in relation to the general problem of search for specific K+/H+ Antiporters in bacteria is discussed.