coli is reversed from the usual orientation of alkaline inside [5] and cannot apparently be used to drive proton uptake into the cell. This is a particular problem when Na+/H+ antiporters are used for alkaline pH homeostasis because, due to the cytotoxicity of Na+[5] it is excluded from the cell and, unlike K+, cannot provide an outwardly-directed driving

force to support an electroneutral exchange. To overcome this, antiporters such as E. coli NhaA [31] and B. subtilis TetL [38], utilise Δψ to catalyse electrogenic Na+/H+ exchange and find more drive net accumulation of H+ to acidify the cytoplasm at alkaline pH in the presence of Na+. Intriguingly, the MdtM homologue MdfA can catalyse both electrogenic and electroneutral transport of drug substrates [39]; however, the component of the PMF that MdfA utilises to drive Na+/H+ or K+/H+ antiport at alkaline pH has not been reported, although it too is likely to be the Δψ. The results of our fluorescence experiments using the Δψ–sensitive probe Barasertib price Oxonol V revealed that MdtM can utilise Δψ as the driving force

at alkaline pH to catalyse an electrogenic Na+(K+)/H+ antiport, i.e., an exchange stoichiometry of >1 H+ per monovalent metal cation (Figure 9). Further evidence to support a physiological role for MdtM in alkaline pH homeostasis was gleaned from HDAC inhibitor estimation of the concentrations of Na+ and K+ required to elicit the half-maximal fluorescence dequench of acridine orange in inverted vesicles (Figure 7). Other transporters that function in bacterial pH homeostasis, such as E. coli NhaB [40], ChaA [12] and MdfA [9], and a sodium-specific

Na+/H+ antiporter from Vibrio parahaemolyticus[41], all possess affinity for their respective metal ion substrate(s) in the general millimolar range. Our values of [Na+]1/2 and [K+]1/2 of 38±6 mM and 32±7 mM, respectively, although not directly related to actual K m values [42], suggest MdtM also possesses relatively low affinity for its cognate metal cations and are therefore consistent with a contributory role for the Na+/H+ and K+/H+ antiporter activities of MdtM in alkaline pH homeostasis. In order to function effectively in pH homeostasis, antiporters must be equipped with sensors of the external and/or cytoplasmic pH that can PIK3C2G transduce the changes in pH into changes in transporter activity [5]. The pH profile of MdtM activity (Figure 7A) suggests that, like other antiporters involved in pH homeostasis, it too is capable of sensing and responding to changes in ionic composition, and provides additional support for our contention that the different antiport functions performed by MdtM are dictated by subtle changes in pH and the type of cation present in the external environment. In our experiments, because MdtM expression from a multicopy plasmid was placed under control of a non-native arabinose-inducible promoter, this suggests an ability to sense pH at the protein level.