Amiloride and harmaline are potent inhibitors of NhaB a Na+/H+ antiporter from Escherichia coli

Pinner, Elhanan; Padan, Etana; Schuldiner, Shimon

doi:10.1016/0014-5793(95)00364-f

Cited by 42 publications

(60 citation statements)

References 29 publications

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“…E. coli strains UT⌬mdfA::kan (R. Edgar and E.B., unpublished data) and EP432 (29) were used for alkaline pH-resistance assays. Cells were transformed with either plasmid pT7-5 or plasmid pT7-5-mdfA-6His (wild type or mutant, as indicated).…”

Section: Methodsmentioning

confidence: 99%

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Alkalitolerance: A biological function for a multidrug transporter in pH homeostasis

Lewinson

Padan

Bibi

2004

Proc. Natl. Acad. Sci. U.S.A.

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MdfA is an Escherichia coli multidrug-resistance transporter. Cells expressing MdfA from a multicopy plasmid exhibit multidrug resistance against a diverse group of toxic compounds. In this article, we show that, in addition to its role in multidrug resistance, MdfA confers extreme alkaline pH resistance and allows the growth of transformed cells under conditions that are close to those used normally by alkaliphiles (up to pH 10) by maintaining a physiological internal pH. MdfA-deleted E. coli cells are sensitive even to mild alkaline conditions, and the wild-type phenotype is restored fully by MdfA expressed from a plasmid. This activity of MdfA requires Na ؉ or K ؉ . Fluorescence studies with inverted membrane vesicles demonstrate that MdfA catalyzes Na ؉ -or K ؉ -dependent proton transport, and experiments with reconstituted proteoliposomes confirm that MdfA is solely responsible for this phenomenon. Studies with multidrug resistance-defective MdfA mutants and competitive transport assays suggest that these activities of MdfA are related. Together, the results demonstrate that a single protein has an unprecedented capacity to turn E. coli from an obligatory neutrophile into an alkalitolerant bacterium, and they suggest a previously uncharacterized physiological role for MdfA in pH homeostasis.MdfA ͉ multidrug transport ͉ sodium proton antiporter ͉ alkaline pH tolerance ͉ Escherichia coli

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Section: Methodsmentioning

confidence: 99%

“…Internal pH measurements were performed essentially as described (31) (29) were transformed with plasmid pUC18 or plasmid pUC18-pARA-mdfA-6His (wild type or mutant, as indicated). Cell growth and preparation of inverted membrane vesicles were performed as described (11).…”

Section: Methodsmentioning

confidence: 99%

Alkalitolerance: A biological function for a multidrug transporter in pH homeostasis

Lewinson

Padan

Bibi

2004

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

111

122

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“…The fast Na+/Li + exchange observed here (Fig. 2) is likely due to the nhaA and/or nhaB antiporters, which then would explain why they confer resistance to Li + [2,3]. The difference in Li+/Na + selectivity between the AST system and the Na+/H + antiporters allows their distinction in vivo, and provides a good basis for future work.…”

Section: Resultsmentioning

confidence: 84%

“…The nhaA and nhaB genes both confer resistance to Li + [2,3], which is toxic for E. coli [4]. Thus, these gene products were considered to extrude Li + as well as Na + from the cells.…”

Section: Introductionmentioning

confidence: 99%

“…Earlier, it was shown that Li +, like Na +, is efficient in dissipating ApH generated by respiration in sub-bacterial vesicles from E. coli [5]. Later, this was found to be due to functioning of the nhaA and nhaB gene products [3]. The nhaB gene product was alone responsible for dissipation of ApH generated by respiration in membrane vesicles from an nhaA gene-deleted mutant, Na + and Li + being equally effective dissipators [6].…”

Section: Introductionmentioning

confidence: 99%

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The respiration‐driven active sodium transport system in E. coli does not function with lithium

1996

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Comparison of respiration-driven active transport of alkali cations from E. coli cells loaded with Na + or Li + showed that Li + could not be expelled from the cells like Na +. K + accumulation, which was fast in Na+-loaded cells, was strongly inhibited in Li+-ioaded cells, despite high membrane potential and respiratory rate. When Li+-loaded cells were placed into medium containing Na + instead of Li +, Li+/Na + exchange took place initially, while K + accumulation was delayed. Only after almost all inside Li + was replaced by Na + did active Na + and K + transport commence. These data confirm that it is a distinct active sodium transport system (AST) with Na+,K+/H + antiporter activity, and not the Na+/H + antiporters, that is responsible for active Na + transport in E. coli Key words: Na + transport; Na+/Li + selectivity; E. coli Materials and methodsThe E. coil strain GR70N was a gift from Dr. R.B. Gennis, University of Illinois. The cells were grown aerobically overnight at 37°C in synthetic salt medium, pH 7.6, containing sodium succinate, and harvested in the middle of the exponential growth phase. Then the cells were loaded with Na + or Li + using the diethanolamine treatment [9]. Loaded cells were concentrated to 3040 mg protein/ml and kept on ice.Transport of alkali cations was initiated by dilution of cell suspensions into aerated, stirred Na + (or Li +) medium, containing 150 mM NaC1 (or LiC1), 50 mM tricine-NaOH (or LiOH), pH 8.5, 5 mM MgSO4, 10 mM KC1, 2 mM dithiotreitol (DTT) and 200 ~tM ubiquinone-I (Q-l). Then the cell suspension was filtered (Millipore, 45 ~tm), the filter was washed with 5 ml 0.5 M mannitol solution, and the content of alkali cations on the filter was measured by flame photometry. Measurements and calculations of electric potential (A~) in EDTA/Tris treated cells, using the tetraphenylphosphonium cation (TPP +) and a TPP+-selective electrode, were performed as described previously [8].

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A single amino acid substitution (Glu134-->Ala) in NhaR1 increases the inducibility by Na+ of the product of nhaA, a Na+/H+ antiporter gene in Escherichia coli.

et al. 1994

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The mutation nhaAup (antup) has now been identified as a Glu134 to Ala substitution in NhaR and designated nhaR1. This was demonstrated by sequence analysis showing that the mutant contains a wild‐type nhaA but nhaR1 instead of nhaR and by the finding that nhaR1 cloned in a plasmid confers the NhaAup phenotype. Na+ (107 mM) increases by 5‐ to 10‐fold the level of nhaA transcripts, similar to the effect on the NhaR‐mediated expression of a nhaA‘‐’lacZ fusion. These results are in agreement with the notion that nhaR is a positive regulator which controls Na(+)‐dependent transcription of nhaA. The promoter region of nhaR and nhaR1 was found to reside within the BglII‐BamHI fragment of the C‐terminal sequences of nhaA. The mutation nhaR1, while increasing dramatically the level of transcription, reduces the requirement for Na+ by 3‐ to 5‐fold both for nhaA transcription and for the nhaR1‐mediated expression of nhaA‘‐’lacZ fusion. NhaR1, like NhaR, binds specifically to the promoter region of nhaA. However, at equal protein concentration NhaR1 binds more DNA and the NhaR1‐DNA complex shows higher mobility than that of NhaR‐DNA, suggesting the existence of two different binding complexes. Yet in this assay the DNA binding pattern of neither NhaR nor NhaR1 was affected by the addition of Na+. The possible relevance of these two DNA‐binding complexes to the Na(+)‐induced NhaR‐mediated expression is discussed.

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Amiloride and harmaline are potent inhibitors of NhaB a Na⁺/H⁺ antiporter from Escherichia coli

Cited by 42 publications

References 29 publications

Alkalitolerance: A biological function for a multidrug transporter in pH homeostasis

Alkalitolerance: A biological function for a multidrug transporter in pH homeostasis

The respiration‐driven active sodium transport system in E. coli does not function with lithium

A single amino acid substitution (Glu134-->Ala) in NhaR1 increases the inducibility by Na+ of the product of nhaA, a Na+/H+ antiporter gene in Escherichia coli.

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