Five pathogenic Candida species were compared in terms of their osmotolerance, tolerance to toxic sodium and lithium cations, and resistance to fluconazole. The species not only differed, in general, in their tolerance to high osmotic pressure (C. albicans and C. parapsilosis being the most osmotolerant) but exhibited distinct sensitivities to toxic sodium and lithium cations, with C. parapsilosis and C. tropicalis being very tolerant but C. krusei and C. dubliniensis sensitive to LiCl. The treatment of both fluconazole-susceptible (C. albicans and C. parapsilosis) and fluconazole-resistant (C. dubliniensis, C. krusei and C. tropicalis) growing cells with subinhibitory concentrations of fluconazole resulted in substantially elevated intracellular Na(+) levels. Using a diS-C3(3) assay, for the first time, to monitor the relative membrane potential (ΔΨ) of Candida cells, we show that the fluconazole treatment of growing cells of all five species results in a substantial hyperpolarization of their plasma membranes, which is responsible for an increased non-specific transport of toxic alkali metal cations and other cationic drugs (e.g., hygromycin B). Thus, the combination of relatively low doses of fluconazole and drugs, whose import into the tested Candida strains is driven by the cell membrane potential, might be especially potent in terms of its ability to inhibit the growth of or even kill various Candida species.
- MeSH
- antifungální látky farmakologie MeSH
- buněčná membrána účinky léků fyziologie MeSH
- Candida účinky léků MeSH
- cytosol chemie MeSH
- flukonazol farmakologie MeSH
- lithium toxicita MeSH
- membránové potenciály účinky léků MeSH
- osmotický tlak MeSH
- sodík toxicita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Three different transport systems exist to accumulate a sufficient amount of potassium cations in yeasts. The most common of these are Trk-type transporters, which are used by all yeast species. Though most yeast species employ two different types of transporters, we only identified one gene encoding a potassium uptake system (Trk-type) in the genome of the highly osmotolerant yeast Zygosaccharomyces rouxii, and our results showed that ZrTrk1 is its major (and probably only) specific potassium uptake system. When expressed in Saccharomyces cerevisiae, the product of the ZrTRK1 gene is localized to the plasma membrane and its presence efficiently complements the phenotypes of S. cerevisiae trk1∆ trk2∆ cells. Deletion of the ZrTRK1 gene resulted in Z. rouxii cells being almost incapable of growth at low K(+) concentrations and it changed some cell physiological parameters in a way that differs from S. cerevisiae. In contrast to S. cerevisiae, Z. rouxii cells without the TRK1 gene contained less potassium than the control cells and their plasma membrane was significantly hyperpolarized compared with those of the parental strain when grown in the presence of 100 mM KCl. On the other hand, subsequent potassium starvation led to a substantial depolarization which is again different from S. cerevisiae. Plasma-membrane hyperpolarization did not prevent the efflux of potassium from Z. rouxii trk1Δ cells during potassium starvation, and the activity of ZrPma1 is less affected by the absence of ZrTRK1 than in S. cerevisiae. The use of a newly constructed Z. rouxii-specific plasmid for the expression of pHluorin showed that the intracellular pH of the Z. rouxii wild type and the trk1∆ mutant is not significantly different. Together with the fact that Z. rouxii cells contain a significantly lower amount of intracellular potassium than identically grown S. cerevisiae cells, our results suggest that this highly osmotolerant yeast species maintain its intracellular pH and potassium homeostasis in way(s) partially distinct from S. cerevisiae.
- MeSH
- biologická adaptace MeSH
- biologický transport MeSH
- buněčná membrána fyziologie MeSH
- delece genu MeSH
- DNA fungální genetika metabolismus MeSH
- draslík metabolismus MeSH
- geny hub * MeSH
- homeostáza MeSH
- homologní rekombinace MeSH
- koncentrace vodíkových iontů MeSH
- membránové potenciály MeSH
- proteiny přenášející kationty genetika metabolismus MeSH
- regulace genové exprese u hub * MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika fyziologie MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie MeSH
- sekvenční seřazení MeSH
- Zygosaccharomyces genetika fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Restraint-based comparative modeling was used for calculation and visualization of the H4-H5-loop of Na+/K+-ATPase from mouse brain (Mus musculus, adult male brain, alpha2-isoform) between the amino acid residues Cys 336 and Arg 758 in the E1 conformation The structure consists of two well separated parts. The N-domain is formed by a seven-stranded antiparallel beta-sheet with two additional beta-strands and five alpha-helices sandwiching it, the P-domain is composed of a typical Rossman fold. The ATP-binding site was found on the N-domain to be identical in both alpha2- and alpha1-isoforms. The phosphorylation Asp 369 residue was found in the central part of the P-domain, located at the C-terminal end of the central beta-sheet. The distance between the alpha-carbon of Phe 475 at the ATP-binding site and the alpha-carbon of Asp 369 at the phosphorylation site is 3.22 nm. A hydrogen bond between the oxygen atom of Asp 369 and the nitrogen atom of Lys 690 was clearly detected and assumed to play a key role in maintaining the proper structure of the phosphorylaton site in E1 conformation.
- MeSH
- adenosintrifosfát metabolismus MeSH
- fosforylace MeSH
- izoenzymy chemie MeSH
- konformace proteinů MeSH
- molekulární modely * MeSH
- molekulární sekvence - údaje MeSH
- mozek enzymologie MeSH
- myši MeSH
- počítačová grafika MeSH
- reprodukovatelnost výsledků MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- sekvenční seřazení MeSH
- sodíko-draslíková ATPasa chemie metabolismus MeSH
- terciární struktura proteinů MeSH
- vazebná místa MeSH
- vodíková vazba MeSH
- zobrazování trojrozměrné MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
The functional expression of the mouse Kir2.1 potassium channel in yeast cells lacking transport systems for potassium and sodium efflux (ena1-4delta nha1delta) resulted in increased cell sensitivity to high external concentrations of potassium. The phenotype depended on the level of Kir2.1 expression and on the external pH. The activity of Kir2.1p in the yeast cells was almost negligible at pH 3.0 and the highest at pH 7.0. Kir2.1p was permeable for both potassium and rubidium cations, but neither sodium nor lithium were transported via the channel. Measurements of the cation contents in cells confirmed the higher concentration of potassium in cells with Kir2.1p. Specific inhibition of the mKir2.1 channel activity by Ba2+ cations was observed. The use of a mutant strain lacking both potassium efflux and uptake transporters (ena1-4delta nha1delta trk1delta trk2delta) enabled the monitoring of channel activity on two levels--the provision of the necessary amount of intracellular K+ in media with low potassium concentrations, and simultaneously, the channel's contribution to cell potassium sensitivity in the presence of high external K+. This combination of mutations proved to be a new, sensitive and practical tool for characterizing the properties of heterologously expressed transporters mediating both the efflux and influx of alkali-metal-cations. Copyright 2005 John Wiley & Sons, Ltd.
- MeSH
- alkalické kovy metabolismus MeSH
- baryum farmakologie MeSH
- draslík metabolismus MeSH
- draslíkové kanály dovnitř usměrňující antagonisté a inhibitory biosyntéza metabolismus MeSH
- fenotyp MeSH
- financování organizované MeSH
- iontový transport MeSH
- Saccharomyces cerevisiae metabolismus růst a vývoj MeSH
- transformace genetická MeSH
