The putative Na+/H+ antiporter of Vibrio cholerae, Vc-NhaP2, mediates the specific K+/H+ exchange in vivo

Abstract

The existence of bacterial K(+)/H(+) antiporters that prevent the overaccumulation of potassium in the cytoplasm was predicted by Peter Mitchell almost 50 years ago. The importance of K(+)/H(+) antiport for bacterial physiology is widely recognized, but its molecular mechanisms remain underinvestigated. Here, we demonstrate that a putative Na(+)/H(+) antiporter, Vc-NhaP2, protects cells of Vibrio cholerae growing at pH 6.0 from high concentrations of external K(+). Resistance of V. cholerae to Na(+) was found to be independent of Vc-NhaP2. When assayed in inside-out membrane vesicles derived from antiporter-deficient Escherichia coli, Vc-NhaP2 catalyzed the electroneutral K(+)(Rb(+))/H(+) exchange with a pH optimum of approximately 7.75 with an apparent K(m) for K(+) of 1.62 mM. In the absence of K(+), it exhibited Na(+)/H(+) antiport, albeit rather weakly. Interestingly, while Vc-NhaP2 cannot exchange Li(+) for protons, elimination of functional Vc-NhaP2 resulted in a significantly higher Li(+) resistance of V. cholerae cells growing at pH 6.0, suggesting the possibility of Vc-NhaP2-mediated Li(+)/K(+) antiport. The peculiar cation specificity of Vc-NhaP2 and the presence of its two additional paralogues in the same genome make this transporter an attractive model for detailed analysis of the structural determinants of the substrate specificity in alkali cation exchangers.

FIGURE 1

Vc-NhaP2 protects V. cholerae from high concentrations of external potassium at acidic pH. The pVc-NhaP2 plasmid, containing Vc-NhaP2, was used to transform V. cholerae strain VcΔNhaP2, bearing deletion of Vc-nhaP2 gene (closed circles). Wild-type cells (closed squares) and VcΔNhaP2 (open circles) were transformed with “empty” pBAD24. Cells were grown aerobically for 18 hours in 96-deep well plates as described in the Experimental Procedures. In all cases the LBB medium, adjusted to the desirable pH, was supplemented with 0.0002% (w/v) arabinose and the indicated concentrations of KCl (A), NaCl (B), or LiCl (C). Growth was measured as the O.D. (600 nm) of the bacterial suspension. The starting O.D. (600 nm) was approximately 0.05 in all cases. Plotted are the averages of three separate experiments, each performed in tripicate. Bars show the standard deviation.

FIGURE 2

Vc-NhaP2 antiport activities in sub-bacterial vesicles. Inside-out membrane vesicles were isolated from TO114 cells transformed with pVc-NhaP2 and assayed with 10 mM of the specified salt in standard choline chloride buffer adjusted to pH 7.5. At the indicated time, respiration-dependent formation of the transmembrane pH gradient was initiated by the addition of 20 mM Tris-D-lactate. After steady-state ΔpH was reached, cation/H⁺ antiporter activity was detected upon the addition of 10 mM KCl (a), 10 mM NaCl (b), 10 mM RbCl (c) and 10 mM LiCl (d). Acridine orange fluorescence is shown in arbitrary units.

FIGURE 3

pH profile of Vc-NhaP2 activity. Inside-out membrane vesicles were isolated from TO114 cells transformed with pVc-NhaP2 or “empty” pBAD24 and assayed with the specified salt in standard choline chloride buffer adjusted to the indicated pH with 50 mM BTP-HCl. In each case, residual non-specific activity measured in “empty” vesicles was substracted from that registered in Vc-NhaP2-containing vesicles and the resulting Vc-NhaP2-dependent activity was plotted as a function of pH. All other conditions as in Fig. 2. Plotted are the averages of six measurements (carried out in duplicate with three separate isolations of vesicles).

FIGURE 4

Determination of kinetic parameters of Vc-NhaP2 in inside-out membrane vesicles isolated from TO114 transformants (inset, double recipricol plot). Measurements were done in standard choline chloride buffer adjusted to pH 7.5 with the final concentration of added KCl (A) and NaCl (B) varying from 0.05 to 50 mM. Each point represents the average of four measurements (carried out in duplicate with two separate isolations of vesicles). Bars show the standard deviation.

FIGURE 5

Na⁺ and Li⁺ compete with K⁺ ions for the Vc-NhaP2 antiporter. Dequenching of acridine orange in response to varying concentrations of KCl was monitored at pH 7.5 in standard choline chloride buffer with or without the indicated concentrations of NaCl or LiCl. Each point represents the average of four measurements (carried out in duplicate with two separate isolations of vesicles). Data are shown in reciprocal coordinates. Bars show the standard deviation.

FIGURE 6

Probing the stoichiometry of Vc-NhaP2. Inside-out membrane vesicles were isolated from TO114 transformants and assayed for ΔΨ at pH 7.5 in sorbitol-based medium devoid of K⁺ and Cl^-. Diethanolamine at 20 mM was added to the experimental mixture 5 min prior to the addition of Oxonol V. At the indicated time, respiration-dependent formation of ΔΨ was initiated by the addition of 20 mM Tris-D-lactate. After steady-state ΔΨ was reached, cation/H⁺ antiport was initiated by the addition of 10 mM K₂SO₄ and 10 mM Na₂SO₄ (as indicated). The protonophore CCCP (traces marked “a”, “c”) or valinomycin in the presence of K⁺ (traces “b”, “d”) was added at the end of each measurement to collapse the generated ΔΨ for the control. The control experiment shown in the two lower panels compares side by side behavior of the electrogenic antiporter, Vc-NhaA (right lower panel, TO114/pBVA vesicles) and Vc-NhaP2 (left lower panel). Fluorescence of Oxonol V is shown in arbitrary units.