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

Potassium is the major monovalent cation of the bacterial cytoplasm. It regulates internal pH, activates many intracellular enzymes and functions as an important osmotic solute (1). However, excessive amounts of internal K+ are detrimental (2–4). Therefore, bacteria tightly regulate their cytoplasmic K+ through the activity of a number of different transport systems (reviewed in (1)). Kdp, Trk and Kup systems import K+ either at the expense of ATP hydrolysis (TrkA and Kdp) or symporting it with a proton (Kup) (5–8). In addition, tetracycline antiporters TetL in Bacillus subtilis and TetK in Staphylococcus aureus that are able to exchange monovalent cations, may contribute to the net K+ uptake (9–11). Export of K+ can be mediated by (a) the glutathione adduct-activated “emergency” KefB/KefC systems of Gram-negative organisms (12); (b) mechanosensitive channels under severe hypoosmotic stress (13–15), although they are thought to play only a minor role in overall K+ homeostasis (1); and (c) the MdfA multidrug resistance transporter, which at external pH >9.0 import protons in exchange for extracellular Na+ or K+ (16). All the above potassium-expelling systems seem to be mobilized only in specific stressful situations. Paradoxically, the identity of system(s) responsible for routine energy-dependent K+ extrusion remains poorly understood. Almost fifty years ago, Peter Mitchell postulated the existence of “housekeeping” K+/H+ and Na+/H+ antiporters, that can directly use the proton motive force to prevent the dangerous over-accumulation of alkali cations (17). Typically, growing bacteria employ a variety of primary proton pumps to maintain a high transmembrane electrical potential difference, ΔΨ (negative inside) over a wide range of external pH. As a result, K+ (or any other monovalent cation), if allowed to equilibrate with the ΔΨ, would accumulate inside the cell at poisonous concentrations. At −120 mV of ΔΨ and a moderate external K+ concentration of 30 mM, at equilibrium the cell would accumulate as much as 3 M K+, a concentration that clearly is beyond the physiological limit. A K+/H+ antiporter would allow H+ expelled by the primary pumps to return into the cytoplasm in exchange for internal K+, thus solving the problem. Although several families of bacterial Na+/H+ antiporters have been identified and studied in great detail (18–22), identification of specific K+/H+ antiporters in bacteria remains elusive. K+/H+ antiport activity as such has been demonstrated in everted membrane vesicles from E. coli a long time ago (23). Some Na+/H+ antiporters, exemplified by well-studied Ec-NhaA and Ec-NhaB (22), are highly discriminative against K+, while others exhibit more or less pronounced K+/H+ exchange as a concomitant activity, such as the multi-subunit Vc-Mrp in Vibrio cholerae (24), or the alkali-activated Aa-NhaP from Alkalimonas amylolytica that transports Na+, K+ and possibly NH4+, but not Li+ (25). Recently, Radchenko and co-authors reported that Vp-NhaP2 from V. parahaemolyticus might be a K+-specific antiporter (4). If confirmed, this would set a valuable precedent, because in spite of the widely recognized importance of K+/H+ antiporters for bacterial ion and pH homeostasis (1), no transporter exclusively specific for K+ has been identified thus far. The authors assayed inside-out vesicles obtained from antiporter-deficient E. coli overexpressing the cloned Vp-NhaP2. The antiporter displayed a rather modest activity with K+ even at its pH optimum of 9.0; in the absence of K+, Na+ seemed to be a substrate as well, albeit poorer than K+ (see Fig. 5B in (4)). Unfortunately, the authors did not examine the effect of Na+ concentration on the Na+/H+ antiport activity. Therefore, definitive conclusions about the specificity of Vp-NhaP2 were hard to make at the moment. Also, one more pressing question remained: would the chromosomal deletion of nhaP2 gene produce a potassium-sensitive phenotype in its native host, V. parahaemolyticus? Inspired by the work of Radchenko and colleagues, we undertook a search for other possible antiporters exclusively transporting K+. In the course of this search we cloned, functionally expressed and examined a homologue of Vp-NhaP2 from Vibrio cholerae O395, Vc-NhaP2, encoded by the open reading frame VC2703. We also engineered and characterized the Vp-NhaP2 chromosomal deletion mutant of V. cholerae. Data presented in this article define Vc-NhaP2 as an electroneutral K+/H+ antiporter, which in vitro is able to catalyze K+/H+, Rb+/H+, Na+/H+ and, possibly, Li+/K+ (but not Li+/H+) exchange, but in situ operates as a Mitchellian K+/H+ antiporter, protecting V. cholerae cells growing at pH 6.0 from high concentrations of K+. The peculiar behavior of Vc-NhaP2 in relation to the general problem of search for specific K+/H+ antiporters in bacteria is discussed.