Abstract
The transmembrane proton gradient (ΔpH) is the primary source of energy exploited by secondary active substrate/H+ antiporters to drive the electroneutral transport of substrates across the Escherichia coli (E. coli) inner membrane. Such electroneutral transport results in no net movement of charges across the membrane. The charge on the transported substrate and the stoichiometry of the exchange reaction, however, can result in an electrogenic reaction which is driven by both the ΔpH and the electrical (∆Ψ) components of the proton electrochemical gradient, resulting in a net movement of electrical charges across the membrane. We have shown that the major facilitator superfamily transporter MdtM - a multidrug efflux protein from E. coli that functions physiologically in protection of bacterial cells against bile salts - imparts bile salt resistance to the bacterial cell by coupling the exchange of external protons (H+) to the efflux of bile salts from the cell interior via an electrogenic antiport reaction (Paul et al., 2014). This protocol describes, using fluorometry, how to detect electrogenic antiport activity of MdtM in inverted membrane vesicles of an antiporter-deficient strain of E. coli TO114 cells by measuring transmembrane ∆Ψ. The method exploits changes that occur in the intensity of the fluorescence signal (quenching and dequenching) of the probe Oxonol V in response to changes in membrane potential due to the MdtM-catalysed sodium cholate/H+ exchange reaction. The protocol can be adapted to detect activity of any secondary active antiporter that couples the electrogenic translocation of H+ across a biological membrane to that of its counter-substrate, and may be used to unmask otherwise camouflaged transport activities and physiological roles.
Keywords: Membrane transport, Exchange, Acridine orange, Fluorescence quenching, Antiporter
Materials and Reagents
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Representative data
Figure 1. Representative data illustrating typical changes in the fluorescence signal of the ΔΨ-sensitive fluorophore Oxonol V in response to MdtM-catalysed electrogenic transport in inverted membrane vesicles. Addition of lactate to energise vesicles results in generation of a respiratory ΔΨ, as evidenced by a rapid quench of the Oxonol V fluorescence signal. (a) Addition of 2.5 mM cholate to inverted vesicles generated from TO114 cells enriched with wild-type MdtM results in a partial depolarization of ΔΨ, represented as a dequenching of the Oxonol V fluorescence, as the ΔΨ was consumed by the MdtM-mediated bile salt/H+ transport reaction. (b) Addition of cholate to negative control vesicles generated from TO114 cells enriched with dysfunctional MdtM D22A results in a small but perceptible dequench arising from residual electrogenic Na+/H+ antiport activity of the mutant. (c) Positive control assay in which Na+ ions are added to inverted vesicles that contain a full complement of electrogenic antiporters in order to measure electrogenic Na+/H+ activity of NhaA transporter. (d) Response of Oxonol V fluorescence to addition of the nigericin. In the presence of K+ ions, this ionophore selectively dissipates ΔpH and converts it into ΔΨ, resulting in a further quench of the fluorescence signal. Valinomycin collapses the ΔΨ. In assays (a, b and c), addition of the protonophore CCCP at the time indicated resulted in almost complete dissipation of ΔΨ. The fluorescence intensity is measured in counts per second (cps). The fluorescence intensity you measure may differ depending upon how your instrument is set up.
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Acknowledgments
This work was supported by BBSRC grant BB/K014226/1 (to CJL). The protocol described above is adapted from one reported previously (Resch et al., 2010).
References
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