Figure 9.

The electrogenicity of MdtM-catalysed Na⁺/H⁺ and K⁺/H⁺ antiport. The electrogenicity of MdtM-catalysed Na⁺/H⁺ and K⁺/H⁺ antiport at alkaline pH was probed by Oxonol V fluorometry of inverted vesicles generated from E. coli TO114 cells transformed with pMdtM (A, C & E) or, as a negative control, pD22A (B & D). Inverted vesicles isolated from BW25113 cells were used as a positive control (F). Respiration-dependent formation of Δψ was initiated by addition of lactate at the time indicated. Once steady-state Δψ was achieved, antiport was initiated by addition of 100 mM Na⁺ gluconate (A & B) or 100 mM K⁺ gluconate (C & D) as indicated. Vesicles were depolarised by addition of CCCP or valinomycin in the presence of K⁺ as indicated. Fluorescence measurements on TO114 inverted vesicles were conducted at either pH 9.0 (for detection of K⁺/H⁺ antiport; panels C & D) or pH 9.25 (for detection of Na⁺/H⁺ antiport; panels A & B), whereas positive control measurements using vesicles derived from BW25113 cells were done at pH 8.5 to ensure detection of the activity of the electrogenic antiporter, NhaA (panel F). The Oxonol V fluorescence is presented as a percentage of the initial fluorescence prior to establishment of the steady-state Δψ. The traces shown are representative of experiments performed in triplicate on two separate preparations of inverted vesicles.