European heart journal, ISSN 1520-765X vol. 1, suppl. K, July 1999
K49 s. : tab., il. ; 30 cm
- MeSH
- Coronary Disease drug therapy MeSH
- Sodium-Hydrogen Exchangers antagonists & inhibitors MeSH
- Publication type
- Congress MeSH
- Conspectus
- Patologie. Klinická medicína
- NML Fields
- kardiologie
- angiologie
Na(+)/H(+) antiporters may recognize all alkali-metal cations as substrates but may transport them selectively. Plasma-membrane Zygosaccharomyces rouxii Sod2-22 antiporter exports Na(+) and Li(+), but not K(+). The molecular basis of this selectivity is unknown. We combined protein structure modeling, site-directed mutagenesis, phenotype analysis and cation efflux measurements to localize and characterize the cation selectivity region. A three-dimensional model of the ZrSod2-22 transmembrane domain was generated based on the X-ray structure of the Escherichia coli NhaA antiporter and primary sequence alignments with homologous yeast antiporters. The model suggested a close proximity of Thr141, Ala179 and Val375 from transmembrane segments 4, 5 and 11, respectively, forming a hydrophobic hole in the putative cation pathway's core. A series of mutagenesis experiments verified the model and showed that structural modifications of the hole resulted in altered cation selectivity and transport activity. The triple ZrSod2-22 mutant T141S-A179T-V375I gained K(+) transport capacity. The point mutation A179T restricted the antiporter substrate specificity to Li(+) and reduced its transport activity, while serine at this position preserved the native cation selectivity. The negative effect of the A179T mutation can be eliminated by introducing a second mutation, T141S or T141A, in the preceding transmembrane domain. Our experimental results confirm that the three residues found through modeling play a central role in the determination of cation selectivity and transport activity in Z. rouxii Na(+)/H(+) antiporter and that the cation selectivity can be modulated by repositioning a single local methyl group.
- MeSH
- Point Mutation MeSH
- Potassium metabolism MeSH
- Fungal Proteins chemistry genetics metabolism MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Cations metabolism MeSH
- Protein Conformation MeSH
- Lithium metabolism MeSH
- Models, Molecular MeSH
- Molecular Sequence Data MeSH
- Sodium-Hydrogen Exchangers chemistry genetics metabolism MeSH
- Amino Acid Sequence MeSH
- Sodium metabolism MeSH
- Substrate Specificity MeSH
- Zygosaccharomyces chemistry genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
There are three different sodium transport systems (Ena1-4p, Nha1p, Nhx1p) in Saccharomyces cerevisiae. The effect of their absence on the tolerance to alkali-metal cations and on the membrane potential was studied. All three sodium transporters were found to participate in the maintenance of Na+, Li+, K+ and Cs+ homeostasis. Measurements of the distribution of a fluorescent potentiometric probe (diS-C3(3) assay) in cell suspensions showed that the lack of all three transporters depolarizes the plasma membrane. The overexpression of the Na+,K+/H+ antiporter Nha1 resulted in the hyperpolarization of the plasma membrane and consequently increased the sensitivity to Cs+, Tl+ and hygromycin B. This is the first evidence that the activity of a Na+,K+/H+ antiporter could play a role in the homeostatic regulation of the plasma membrane potential in yeast cells.
- MeSH
- Potassium metabolism MeSH
- Financing, Organized MeSH
- Hydrogen-Ion Concentration MeSH
- Membrane Potentials MeSH
- Membrane Proteins physiology MeSH
- Sodium-Hydrogen Exchangers physiology MeSH
- Cation Transport Proteins physiology MeSH
- Saccharomyces cerevisiae Proteins physiology MeSH
- Saccharomyces cerevisiae physiology growth & development MeSH
- Sodium metabolism MeSH
The human Na+ /H+ antiporter NHA2 (SLC9B2) transports Na+ or Li+ across the plasma membrane in exchange for protons, and is implicated in various pathologies. It is a 537 amino acids protein with an 82 residues long hydrophilic cytoplasmic N-terminus followed by a transmembrane part comprising 14 transmembrane helices. We optimized the functional expression of HsNHA2 in the plasma membrane of a salt-sensitive Saccharomyces cerevisiae strain and characterized in vivo a set of mutated or truncated versions of HsNHA2 in terms of their substrate specificity, transport activity, localization, and protein stability. We identified a highly conserved proline 246, located in the core of the protein, as being crucial for ion selectivity. The replacement of P246 with serine or threonine resulted in antiporters with altered substrate specificity that were not only highly active at acidic pH 4.0 (like the native antiporter), but also at neutral pH. P246T/S versions also exhibited increased resistance to the HsNHA2-specific inhibitor phloretin. We experimentally proved that a putative salt bridge between E215 and R432 is important for antiporter function, but also structural integrity. Truncations of the first 50-70 residues of the N-terminus doubled the transport activity of HsNHA2, while changes in the charge at positions E47, E56, K57, or K58 decreased the antiporter's transport activity. Thus, the hydrophilic N-terminal part of the protein appears to allosterically auto-inhibit cation transport of HsNHA2. Our data also show this in vivo approach to be useful for a rapid screening of SNP's effect on HsNHA2 activity.
Yeasts tightly regulate their intracellular concentrations of alkali metal cations. In Saccharomyces cerevisiae, the Nha1 Na(+) /H(+) -antiporter and Ena1 Na(+) -ATPase, mediate the efflux of toxic sodium and surplus potassium. We report the characterization of Candida glabrata CgCnh1 and CgEna1 homologues. Their substrate specificity and transport properties were compared upon expression in S. cerevisiae, and their function characterized directly in C. glabrata. The CgCnh1 antiporter and the CgEna1 ATPase transport both potassium and sodium when expressed in S. cerevisiae. CgEna1p fully complements the lack of S. cerevisiae own Na(+) -ATPases but the activity of the CgCnh1 antiporter is lower than that of ScNha1p. Candida glabrata deletion mutants and analyses of their phenotypes revealed that though both transporters have a broad substrate specificity, their function in C. glabrata cells is not the same. Their differing physiological roles are also reflected in their regulation of expression, CgENA1 is highly upregulated by an increased osmotic pressure or sodium concentration, whereas CgCNH1 is expressed constitutively. The Cnh1 antiporter is involved in the regulation of potassium content and the Ena1 ATPase in sodium detoxification of C. glabrata cells. This situation differs from S. cerevisiae, where the Nha1 antiporter and Ena ATPases both participate together in Na(+) detoxification and in the regulation of K(+) homeostasis.
- MeSH
- Candida glabrata genetics metabolism physiology MeSH
- Gene Deletion MeSH
- Potassium metabolism MeSH
- Fungal Proteins genetics metabolism MeSH
- Homeostasis MeSH
- Cations metabolism MeSH
- Sodium-Hydrogen Exchangers genetics metabolism MeSH
- Gene Expression Regulation, Fungal MeSH
- Saccharomyces cerevisiae metabolism MeSH
- Sodium metabolism MeSH
- Substrate Specificity MeSH
- Genetic Complementation Test MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
The physiological role of Candida albicans Cnh1, a member of the Na+/H+ antiporter family, was characterized. Though CaCnh1p had broad substrate specificity and mediated efflux of at least four alkali metal cations upon heterologous expression in Saccharomyces cerevisiae, its presence in C. albicans cells was important especially for potassium homeostasis. In C. albicans, CaCnh1p tagged with GFP was localized in the plasma membrane of cells growing as both yeasts and hyphae. Deletion of CNH1 alleles did not affect tolerance to NaCl, LiCl or CsCl, but resulted in increased sensitivity to high external concentrations of KCl and RbCl. The potassium and rubidium tolerance of a cnh1 homozygous mutant was fully restored by reintegration of CNH1 into the genome. The higher sensitivity of the cnh1/cnh1 mutant to external KCl was caused by a lower K+ efflux from these cells. Together, the functional characterization of the CaCnh1 antiporter in C. albicans revealed that this antiporter plays a significant role in C. albicans physiology. It ensures potassium and rubidium tolerance and participates in the regulation of intracellular potassium content of C. albicans cells.
- MeSH
- Anti-Bacterial Agents pharmacology MeSH
- Cell Membrane chemistry MeSH
- Candida albicans metabolism MeSH
- Cesium pharmacology MeSH
- Potassium Chloride pharmacology MeSH
- Lithium Chloride pharmacology MeSH
- Sodium Chloride pharmacology MeSH
- Chlorides pharmacology MeSH
- Gene Deletion MeSH
- Potassium metabolism MeSH
- Gene Expression MeSH
- Financing, Organized MeSH
- Drug Resistance, Fungal MeSH
- Fungal Proteins analysis genetics metabolism MeSH
- Homeostasis MeSH
- Hyphae chemistry MeSH
- Cloning, Molecular MeSH
- Yeasts chemistry MeSH
- Sodium-Hydrogen Exchangers analysis genetics metabolism MeSH
- Rubidium pharmacology MeSH
- Saccharomyces cerevisiae genetics metabolism MeSH
- Substrate Specificity MeSH
- Genetic Complementation Test MeSH
Yarrowia lipolytica plasma-membrane Na+/H+ antiporter, encoded by the YlNHA2 gene, is a very efficient exporter of surplus sodium from the cytosol. Its heterologous expression in Saccharomyces cerevisiae wild-type laboratory strains increased their sodium tolerance more efficiently than the expression of ZrSod2-22 antiporter from the osmotolerant yeast Zygosaccharomvces rouxii.
- MeSH
- Antiporters MeSH
- Sodium Chloride chemistry MeSH
- Financing, Organized utilization MeSH
- Threshold Limit Values MeSH
- Saccharomyces cerevisiae cytology chemistry metabolism MeSH
- Sodium chemistry metabolism adverse effects MeSH
- Cell Survival MeSH
- Yarrowia cytology chemistry metabolism MeSH
- Zygosaccharomyces cytology chemistry metabolism MeSH
Na+/H+ antiporters, integral membrane proteins that exchange protons for alkali metal cations, play multiple roles in probably all living organisms (preventing cells from excessive amounts of alkali metal cations, regulating intracellular pH and cell volume). In this work, we studied the functionality of rat plasma membrane NHE1-3 exchangers upon their heterologous expression in alkali-metal-cation sensitive Saccharomyces cerevisiae, and searched for conditions that would increase their level in the plasma membrane and improve their functionality. Though three tested exchangers were partially localized to the plasma membrane (and two of them (NHE2 and NHE3) in an active form), the bulk of the synthesized proteins were arrested along the secretory pathway, mainly in the ER. To increase the level of exchangers in the yeast plasma membrane several approaches (truncation of C-terminal regulatory sequences, expression in mutant yeast strains, construction of rat/yeast protein chimeras, various growth conditions and chemical chaperones) were tested. The only increase in the amount of NHE exchangers in the plasma membrane was obtained upon expression in a strain with the npi1 mutation, which significantly lowers the level of Rsp5 ubiquitin ligase in cells. This mutation helped to stabilize proteins in the plasma membrane.
- MeSH
- Phenotype MeSH
- Financing, Organized MeSH
- Glycerol pharmacology MeSH
- Cloning, Molecular MeSH
- Rats MeSH
- Humans MeSH
- Mutant Chimeric Proteins physiology MeSH
- Sodium-Hydrogen Exchangers biosynthesis genetics MeSH
- Recombinant Proteins biosynthesis MeSH
- Saccharomyces cerevisiae genetics metabolism drug effects MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Animals MeSH
