Sodium-proton antiport in isolated membrane vesicles of Escherichia coli

Schuldiner, Shimon; Fishkes, H

doi:10.1021/bi00597a023

Cited by 176 publications

(101 citation statements)

References 44 publications

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“…A reversal of the fluorescence level (dequenching) indicates that protons are exiting the vesicles in antiport with either Na ϩ or Li ϩ . As shown previously the end level of dequenching is a good estimate of the antiporter activity (30) and the concentration of the ion that gives half-maximal dequenching is a good estimate of the apparent K m of the antiporter (30,31). The concentration range of the cations tested was 0.01-100 mM at the indicated pH values and the apparent K m values were calculated by linear regression of a LineweaverBurk plot.…”

Section: Methodsmentioning

confidence: 79%

Transmembrane Segment II of NhaA Na+/H+ Antiporter Lines the Cation Passage, and Asp65 Is Critical for pH Activation of the Antiporter

Herz

Rimon

Olkhova

et al. 2010

Journal of Biological Chemistry

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The crystal structure of Escherichia coli NhaA determined at pH 4 has provided insights into the mechanism of activity of a pH-regulated Na ؉ /H ؉ antiporter. However, becauseNhaA is activated at physiological pH (pH 5.5-8.5), many questions related to the active state of NhaA have remained elusive. Our experimental results at physiological pH and computational analyses reveal that amino acid residues in transmembrane segment II contribute to the cation pathway of NhaA and its pH regulation: 1) transmembrane segment II is a highly conserved helix and the conserved amino acid residues are located on one side of the helix facing either the cytoplasmic or periplasmic funnels of NhaA structure. 2) Cys replacements of the conserved residues and measuring their antiporter activity in everted membrane vesicles showed that D65C, L67C, E78C, and E82C increased the apparent K m to Na ؉ and Li Sodium proton antiporters are ubiquitous membrane proteins found in the cytoplasmic and organelle membranes of cells of many different origins, including plants, animals, and microorganisms. They are involved in cell energetics and play primary roles in the regulation of intracellular pH, cellular Na ϩ content, and cell volume (see reviews in Refs. 1-3).

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Section: Methodsmentioning

confidence: 79%

Transmembrane Segment II of NhaA Na+/H+ Antiporter Lines the Cation Passage, and Asp65 Is Critical for pH Activation of the Antiporter

Herz

Rimon

Olkhova

et al. 2010

Journal of Biological Chemistry

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show abstract

“…The activity of NhaB is independent of pH [200,202], but the actual Na+/H ÷ stoichiometry of the exchange reaction is unknown at present. Since the variations in the apparent Na+/H + stoichiometry in membrane vesicles and cells, expressing both NhaA and NhaB [203,204], parallel the pH dependence of NhaA [196,197,199], an electroneutral exchange by NhaB would be most consistent with the observed stoichiometries.…”

Section: V-f Na + / H ÷ Antiporter (Nhaa) Of E Colimentioning

confidence: 79%