Moreover, this inhibition was titratable; addition of increasing concentrations of Na+ resulted in an increasing inhibition of EtBr efflux. Addition of choline chloride had no measurable effect on EtBr efflux (data not shown), thereby establishing that the inhibition of EtBr efflux by NaCl was due solely to Na+ ions. Together, the results of the whole cell transport assays suggest that EtBr and Na+ utilise the same binding site and/or translocation

pathway in MdtM. Indeed, in the closely related MdtM homolog MdfA, the multidrug and Na+ cation translocation pathways Akt inhibitor overlap [9]. Figure 5 Whole cell ethidium bromide transport assays performed in the presence of different concentrations of NaCl. Representative traces of the efflux of EtBr from cells expressing wild-type MdtM in the presence of 0 mM (A), 20 mM (B), 50 mM (C) and 100 mM (D) NaCl. EtBr efflux was monitored continuously by measuring fluorescence emission at 600 nm upon excitation at 545 nm. UTL2 cells that expressed the MdtM D22A mutant in the absence of added NaCl www.selleckchem.com/products/GDC-0449.html were used as a control (E). Cells loaded with EtBr were energised by addition of glucose (as indicated by the first arrow) and efflux of EtBr was monitored for 800 s. CCCP (100 μM) was added (as indicated by the second arrow) to abolish active transport. Fluorescence intensity was measured in counts per second (cps).

MdtM catalyses K+/H+ and Na+/H+ exchange activities The growth assay and whole cell EtBr efflux data implied that MdtM-catalysed K+/H+ and Na+/H+ antiport activities underpinned alkalitolerance. To examine if MdtM mediated the exchange of K+ and Na+ for protons, we measured the changes in

luminal pH of inverted membrane vesicles generated from antiporter-deficient TO114 cells [26] that overexpressed wild-type MdtM by monitoring the fluorescence dequenching of acridine orange upon addition of Na+ Cobimetinib mouse gluconate or K+ gluconate to the transport assay buffer at the indicated alkaline pH values (Figure 6). Inverted vesicles prepared from TO114 cells that overproduced dysfunctional MdtM D22A mutant were used as controls. Figure 6 Cation-driven proton translocation by MdtM. Cation-driven proton translocation by MdtM at alkaline pH was measured by the fluorescence dequenching of acridine orange upon addition of Na+ gluconate (A) or K+ gluconate (B) to inverted vesicles derived from antiporter-deficient E. coli TO114 cells that overexpressed recombinant wild-type MdtM (black traces) or the dysfunctional MdtM D22A mutant (grey traces). Respiration-dependent generation of ΔpH (acid inside) was established by addition of lactate as indicated and once the fluorescence quench of acridine orange reached a steady state, Na+ gluconate or K+ gluconate was added to a final concentration of 100 mM. Addition of 100 μM CCCP at the time indicated was used to completely dissipate ΔpH.