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Vc-NhaD is a Na(+)/H(+) antiporter from Vibrio cholerae with a sharp maximum of activity at pH approximately 8.0. NhaD homologues are present in many bacteria as well as in higher plants. However, very little is known about structure-function relations in NhaD-type antiporters. In this work 14 conserved polar residues associated with putative transmembrane segments of Vc-NhaD have been screened for their possible role in the ion translocation and pH regulation of Vc-NhaD. Substitutions S150A, D154G, N155A, N189A, D199A, T201A, T202A, S389A, N394G, S428A, and S431A completely abolished the Vc-NhaD-mediated Na(+)-dependent H(+) transfer in inside-out membrane vesicles. Substitutions T157A and S428A caused a significant increase of apparent K(m) values for alkali cations, with the K(m) for Li(+) elevated more than that for Na(+), indicating that Thr-157 and Ser-428 are involved in alkali cation binding/translocation. Of six conserved His residues, mutation of only His-93 and His-210 affected the Na(+)(Li(+))/H(+) antiport, resulting in an acidic shift of its pH profile, whereas H93A also caused a 7-fold increase of apparent K(m) for Na(+) without affecting the K(m) for Li(+). These data suggest that side chains of His-93 and His-210 are involved in proton binding and that His-93 also contributes to the binding of Na ions during the catalytic cycle. These 15 residues are clustered in three distinct groups, two located at opposite sides of the membrane, presumably facilitating the access of substrate ions to the third group, a putative catalytic site in the middle of lipid bilayer. The distribution of these key residues in Vc-NhaD molecule also suggests that transmembrane segments IV, V, VI, X, XI, and XII are situated close to one another, creating a transmembrane relay of charged/polar residues involved in the attraction, coordination, and translocation of transported cations.

The Vc-NhaD is an Na + /H + antiporter from Vibrio cholerae belonging to a new family of bacterial Na + /H + antiporters, the NhaD family. In the present work we mutagenized five conserved Asp and Glu residues and one conserved Thr residue to Ala in order to identify amino acids that are critical for the antiport activity. All mutations fall into two distinct groups: (i) four variants, Glu 100 Ala, Glu 251 Ala, Glu 342 Ala, and Asp 393 Ala, did not abolish antiport activity but shifted the pH optimum to more alkaline pH, and (ii) variants Asp 344 Ala, Asp 344 Asn, and Thr 345 Ala caused a complete loss of both Na + /H + and Li + /H + antiport activity whereas the Asp 344 Glu variant exhibited reduced Na + /H + and Li + /H + antiport activity. This is the first mutational analysis of the antiporter of NhaD type and the first demonstration of Thr residue being indispensable for Na + /H + antiport. We discuss the possible role of Asp 344 and Thr 345 in the functioning of Vc-NhaD. D 2002 Elsevier Science B.V. All rights reserved.

The Escherichia coli mutant ΔnhaAΔnhaB (EP432), which lacks the two specific Na+/H+ antiporter genes, is incapable of efficiently excreting Na+. Accordingly at low K+ (6 mM) medium, its intracellular Na+ concentration is only slightly lower (1.5-2×) than the extracellular concentration (50 mM), explaining the high sensitivity to Na+ (≥30 mM) of the mutant. This Na+ sensitivity is shown to be a powerful selection for spontaneous second-site suppressor mutations that allow growth on high Na+ (≤0.6 M) with a rate similar to that of the wild type. One such mutation, MH1, maps at 25.7 min on the E. coli chromosome. It confers Na+ but not Li+ resistance upon ΔnhaAΔnhaB cells and exposes a Na+-excreting capacity, maintaining a Na+ gradient of about 8-10 (at 50 mM extracellular Na+), which is similar to that of the wild type. Although lower, Na+ excretion capacity is also observed in the ΔnhaAΔnhaB mutant when grown in medium containing higher K+ (70 mM). This capacity is accompanied with a shift in the sensitivity of the mutant to higher Na+ concentrations (≥300 mM). Whereas Na+ excretion by a wild type carrying Δunc is uncoupler sensitive, that of MH1Δunc is dependent on respiration in an uncoupler-insensitive fashion. It is concluded that under some conditions (high K+ in the medium or in MH1-like mutants), a primary pump driven by respiration is responsible for Na+ extrusion when the Na+/H+ antiporters are not active.

Mechanisms of Na+ transport into the inside-out subcellular vesicles of alkalo- and halotolerant Bacillus FTU and of Escherichia coli grown at different pH have been studied. Both microorganisms growing at pH 7.5 are shown to possess a system of the respiration-dependent Na+ transport which (i) is inhibited by protonophorous uncoupler, by ΔpH-discharging agent diethylammonium (DEA) acetate, by micromolar cyanide arresting the H+-motive respiratory chain, and by amiloride, and (ii) is resistant to the Na+/H+ antiporter monensin and to Ag+, inhibitor of the Na+-motive respiratory chain. Growth at pH 8.6 strongly changes the activator and inhibitor pattern. Now (1) protonophore stimulates the Na+ transport, (2) DEA acetate is without effect in the absence of protonophore and is stimulating in its presence, (3) amiloride and low cyanide are ineffective, (4) monensin and Ag+ completely arrest the Na+ accumulation in the vesicles. Independent of pH of the growth medium, (a) valinomycin is stimulatory for the Na+ transport, (b) Na+ ionophore ETH 157 is inhibitory and, (c) Na+ transport can be supported by NADH → fumarate as well as by ascorbate (TMPD) → O2 electron transfers. Growth at alkaline pH results in the appearance of ascorbate (TMPD) oxidation resistant to low and sensitive to high cyanide concentrations. These relationships are in agreement with the concept (Skulachev, V.P. (1984) Trends Biochem. Sci. 9, 483–485) that adaptation to alkaline conditions in bacteria growing in the high [Na+] media causes substitution of Na+ for H+ as a coupling ion. The obtained data indicate that under alkaline conditions, Na+ can be pumped from the cell by the Na+-motive respiratory chain with neither H+-motive respiration nor the Na+/H+ antiporter involved. In the Na+-motive respiratory chain of Bac. FTU or E. coli, two Na+ pumps are localized, one in its initial and the other in its terminal spans.