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 (original) (raw)
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The Journal of biological chemistry, 1998
pH controls the activity of the NhaA Na ؉ /H ؉ antiporter of Escherichia coli. In the present work we show that replacement of glycine 338 of NhaA with serine (G338S) alleviates the pH control of the antiporter. Monitoring Na ؉ -dependent collapse of ⌬pH, to assess antiporter activity in isolated membrane vesicles, shows that the mutant protein is practically independent of pH, between pH 7 and 9, and even at pH 6 is 70% active. Similarly the purified reconstituted mutant protein catalyzes pH-independent passive efflux of 22 Na from proteoliposomes as well as ⌬pH-driven influx. Whereas the native NhaA in isolated membrane vesicles is exposed to digestion by trypsin only above pH 7, the mutated protein is degraded already at pH 6.5. ⌬nhaA⌬nhaB cells transformed with a plasmid encoding the pH-independent antiporter are sensitive to Na ؉ but not to K ؉ at alkaline pH, while growing in the presence of both ions at neutral pH. Several possibilities that could explain the Na ؉ sensitivity of the mutant at alkaline pH were excluded; Western analysis and measurement of Na ؉ /H ؉ antiporter activity in membrane vesicles, isolated from cells shifted to the non-permissive growth conditions, showed neither reduced expression of G338S-NhaA nor defective activity. The finding that the mutated protein is electrogenic led to the retraction of the idea that the protein is active in vitro but not in vivo at alkaline pH, when only ⌬ exists in the cells. The Na ؉ concentration needed for half-maximal activity of G338S in isolated everted membrane vesicles is similar to that of the wild type. Therefore an increase in intracellular Na ؉ due to a reduced antiporter affinity could not explain the results. It is suggested that the loss of growth at alkaline pH in the presence of Na ؉ is due to the loss of the pH control of the mutated NhaA. Indeed, in the four mutations suppressing G338S phenotype, growth at alkaline pH was restored together with the pH regulation of NhaA. Three of the four suppressor mutations cluster in helix IV, whereas the original mutation is in helix XI, suggesting that the two helixes interact.
Biochimie, 1986
The Na÷/H + antiport is present in the plasma membrane of virtually all vertebrate cells and it plays a central role in cell homeostasis. The pharmacological properties and the characteristics of the interaction of extracellular Na +, Li +, H + and of intracellular H + with the Na+/H + antiport are reviewed herein. The kinetic properties of the system are shown to be essential for defining its four main physiological functions: (1) transepithelial ion transport, (2) control of the pH i, (3) control of the intracellular Na ÷ concentration, and (4) control of the cell volume.
The Journal of general physiology, 1995
Na+/H+ exchange in vertebrates is thought to be electroneutral and insensitive to the membrane voltage. This basic concept has been challenged by recent reports of antiport-associated currents in the turtle colon epithelium (Post and Dawson, 1992, 1994). To determine the electrogenicity of mammalian antiporters, we used the whole-cell patch clamp technique combined with microfluorimetric measurements of intracellular pH (pHi). In murine macrophages, which were found by RT-PCR to express the NHE-1 isoform of the antiporter, reverse (intracellular Na(+)-driven) Na+/H+ exchange caused a cytosolic acidification and activated an outward current, whereas forward (extracellular Na(+)-driven) exchange produced a cytosolic alkalinization and reduced a basal outward current. The currents mirrored the changes in pHi, were strictly dependent on the presence of a Na+ gradient and were reversibly blocked by amiloride. However, the currents were seemingly not carried by the Na+/H+ exchanger itself...
Insight into the direct interaction of Na+ with NhaA and mechanistic implications
Scientific Reports, 2021
Na+/H+ antiporters comprise a family of membrane proteins evolutionarily conserved in all kingdoms of life that are essential in cellular ion homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystals provided insight in the structure of this molecular machine. However, structural data revealing the composition of the binding site for Na+ (or its surrogate Li+) is missing, representing a bottleneck in our understanding of the correlation between the structure and function of NhaA. Here, by adapting the scintillation proximity assay (SPA) for direct determination of Na+ binding to NhaA, we revealed that (i) NhaA is well adapted as the main antiporter for Na+ homeostasis in Escherichia coli and possibly in other bacteria as the cytoplasmic Na+ concentration is similar to the Na+ binding affinity of NhaA, (ii) experimental conditions affect NhaA-mediated catio...
Responsiveness of mutants of NHE1 isoform of Na+/H+ antiport to osmotic stress
American Journal of Physiology-Cell Physiology, 1995
Hypertonic activation of NHE1, the ubiquitous Na+/H+ exchanger, plays a central role in cell volume regulation, yet little is known about the underlying mechanism. We probed the osmotic responsiveness of full-length and truncated constructs of NHE1 transfected into cells lacking endogenous antiport activity. The hypertonic stimulation of NHE1 was preserved after heterologous transfection of the full-length NHE1 or of constructs truncated at positions 698 or 703. In contrast, mutants truncated at position 635 (delta 635) failed to respond to osmotic challenge. Transfectants (delta 635) behaved as if constitutively activated, having a permanently elevated cytosolic pH (pHi) under isotonic, unstimulated conditions. The delta 635 mutant displayed H+ binding with high affinity and low cooperativity. Constructs delta 582 or delta 566 had a reduced H+ sensitivity and were therefore inactive at resting pHi. Such cells were unresponsive to osmotic stress near physiological pHi but could be a...