Chemical modifications of the Na+– H+ antiport in Escherichia coli membrane vesicles

Damiano, Evelyne; Bassilana, Martine; Leblanc, Gérard

doi:10.1111/j.1432-1033.1985.tb08823.x

Cited by 29 publications

(8 citation statements)

References 23 publications

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“…In the present work we found that the three cysteine residues of NhaA are not essential in the mechanism of the antiporter, confirming previous findings on the lack of inhibition by SH reagents (17). The functional antiporter devoid of cysteinyl residues has provided the basis to design mutants of NhaA in which a suggested important residue such as His-225 is replaced with cysteinyl residue which can then be reacted specifically with sulfhydryl reagents.…”

supporting

confidence: 91%

Histidine 225, a Residue of the NhaA-Na+/H+ Antiporter of Escherichia coli Is Exposed and Faces the Cell Exterior

Olami

Rimon

Gerchman

et al. 1997

Journal of Biological Chemistry

105

137

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Cysteine residues were found nonessential in the mechanism of the NhaA antiporter activity of Escherichia coli. The functional C-less NhaA has provided the groundwork to study further histidine 225 of NhaA which has previously been suggested to play an important role in the activation of NhaA at alkaline pH (Rimon, A., Gerchman, Y., Olami, Y., Schuldiner, S. and Padan, E. (1995) J. Biol. Chem. 270, 26813-26817). C-less H225C was constructed and shown to possess an antiporter activity 60% of that of C-less antiporter and a pH profile similar to that of both the C-less or wild-type antiporters. Remarkably, whereas neither the wild-type nor the C-less antiporters were affected by N-ethylmaleimide, C-less H225C was inhibited by this reagent. To determine the degree of alkylation of the antiporter protein by N-ethylmaleimide, antiporter derivatives tagged at their C termini with six histidines residues were constructed. Alkylation of C-less H225C was measured by labeling of everted membrane vesicles with [14 C]N-ethylmaleimide, affinity purification of the His-tagged antiporter, and determination of the radioactivity of the purified protein. This assay showed that H225C is alkylated to a much higher level than any of the native cysteinyl residues of NhaA reaching saturation at alkyl/ NhaA stoichiometry of 1. The wild-type derivative showed at least 10-fold less alkylation even at higher concentrations, suggesting that H225C resides in a domain that is much more exposed to N-ethylmaleimide than the native cysteinyl residues of NhaA.Since H225C residues both in right-side out and inside-out membrane vesicles were quantitatively alkylated by N-ethylmaleimide, this assay was used to determine the accessibility of H225C to other SH reagents by titrating the H225C left free to react with N-ethylmaleimide, following exposure of the membranes to the reagents. Furthermore, since membrane-impermeant probes can react with residues in membrane-embedded protein only if accessible to the medium containing the reagent, the assay was used to determine the membrane topology of H225C.As expected for a membrane-permeant probe, p-chloromercuribenzoate reacted with H225C as efficiently as N-ethylmaleimide in both membrane orientations. Similar results were obtained with methanethiosulfonate ethylammonium supporting the recent observations that this probe is membrane-permeant. On the other hand, both membrane-impermeant reagents p-chloromercuribenzosulfonate and methanethiosulfonate ethyl-trimethyl ammonium bromide reacted with H225C 10-fold more in right-side out than in inside-out vesicles, and p-chloromercuribenzosulfonate also blocked completely the H225C in intact cells. These results strongly suggest that H225C is exposed at the periplasmic face of the membrane.

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supporting

confidence: 91%

Histidine 225, a Residue of the NhaA-Na+/H+ Antiporter of Escherichia coli Is Exposed and Faces the Cell Exterior

Olami

Rimon

Gerchman

et al. 1997

Journal of Biological Chemistry

105

137

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“…Our data are compatible with previously published reports showing an irreversible inhibition of the Na+/H + exchanger in rabbit renal brush-border membrane vesicles by carbodi-imides [10,29,33,45]. In contrast, the Na+/H + exchangers in E. coli [17] and thymic lymphocytes [23] were not sensitive to carbodiimides. It needs to be clarified whether these differences reflect different types of antiporters.…”

Section: Irreversible Inhibitionsupporting

confidence: 92%

Identification of the renal Na+/H+ exchanger with N,N′-dicyclohexylcarbodiimide (DCCD) and amiloride analogues

1986

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Dicyclohexylcarbodiimide (DCCD) and the 5-ethyl-isopropyl-6-bromo-derivative of amiloride (Br-EIPA) have been used as affinity and photoaffinity labels of the Na+/H+ exchanger in rat renal brush-border membranes. Intravesicular acidification by the Na+/H+ exchanger was irreversibly inhibited after incubation of vesicles for 30 min with DCCD. The substrate of the antiporter, Na+, and the competitive inhibitor, amiloride, protected from irreversible inhibition. The Na+-dependent transport systems for sulfate, dicarboxylates, and neutral, acidic, and basic amino acids were inhibited by DCCD, but not protected by amiloride. An irreversible inhibition of Na+/H+ exchange was also observed when brush-border membrane vesicles were irradiated in the presence of Br-EIPA. Na+ and Li+ protected. [14C]-DCCD was mostly incorporated into three brush-border membrane polypeptides with apparent molecular weights of 88,000, 65,000 and 51,000. Na+ did not protect but rather enhanced labeling. In contrast, amiloride effectively decreased the labeling of the 65,000 molecular weight polypeptide. In basolateral membrane vesicles one band was highly labeled by [14C]DCCD that was identified as the alpha-subunit of the Na+,K+-ATPase. [14C]-Br-EIPA was mainly incorporated into a brush-border membrane polypeptide with apparent molecular weight of 65,000. Na+ decreased the labeling of this protein. Similar to the Na+/H+ exchanger this Na+-protectable band was absent in basolateral membrane vesicles. We conclude that a membrane protein with an apparent molecular weight of 65,000 is involved in rat renal Na+/H+ exchange.

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“…Histidines (pK 6.0 in solution) have been implicated in the mechanism of H+ transport in the lactose carrier (18,19), in the photosynthetic reaction center (20), and also in the Na+/H+ antiporter activity of E. coli (21).…”

mentioning

confidence: 99%

Histidine-226 is part of the pH sensor of NhaA, a Na+/H+ antiporter in Escherichia coli.

Gerchman

Olami

Rimon

et al. 1993

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

133

137

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The nhaA gene of Escherichia coli, which encodes a pH-activated Na+/H+ antiporter, has been modified; six of its eight histidine codons were mutated to arginine codons by site-directed mutagenesis, yielding the mutations H254R-H257R (a double mutant), H226R, H39R, H244R, and H319R. In addition a deletion (delta nhaA1-14) lacking the remaining two histidines, His-3 and His-5, has been constructed. By comparing the phenotypes conferred by plasmids bearing the various mutations to the phenotype of the wild type upon transformation of strains NM81 (delta nhaA) or EP432 (delta nhaA, delta nhaB) we found that none of the NhaA histidines are essential for the Na+/H+ antiporter activity of the NhaA protein. However, the replacement of His-226 by Arg markedly changes the pH dependence of the antiporter. All mutants except H226R confer to NM81 and EP432 Na+ resistance up to pH 8.5 as well as Li+ resistance. Cells bearing H226R are resistant to Li+ and to Na+ at neutral pH, but they become sensitive to Na+ above pH 7.5. Analysis of the Na+/H+ antiporter activity of membrane vesicles derived from H226R cells shows that the mutated protein is activated by pH to the same extent as the wild type. However, whereas the activation of the wild-type NhaA occurs between pH 7 and pH 8, that of H226R antiporter occurs between pH 6.5 and pH 7.5. Furthermore, while the wild-type antiporter remains almost fully active at least up to pH 8.5, H226R is reversibly inactivated above pH 7.5, reaching 10-20% of the maximal activity at pH 8.5. We suggest that His-226 is part of a pH-sensitive site that regulates the activity of NhaA.

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Chemical modifications of the Na⁺– H⁺ antiport in Escherichia coli membrane vesicles

Cited by 29 publications

References 23 publications

Histidine 225, a Residue of the NhaA-Na+/H+ Antiporter of Escherichia coli Is Exposed and Faces the Cell Exterior

Histidine 225, a Residue of the NhaA-Na+/H+ Antiporter of Escherichia coli Is Exposed and Faces the Cell Exterior

Identification of the renal Na+/H+ exchanger with N,N′-dicyclohexylcarbodiimide (DCCD) and amiloride analogues

Histidine-226 is part of the pH sensor of NhaA, a Na+/H+ antiporter in Escherichia coli.

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