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Fig. 5. Representation of the CQ-associated H+ leak observed in CQR strains. Normal resting pHDV reflects a balance between the inward H+ pumping of the V-type H+-ATPase and the outward leak of H+ from the DV, via as yet uncharacterised leak pathways (indicated by the boxed question mark). On inhibition of the V-type H+-ATPase with concanamycin A, efflux of H+ via the leak pathways results in DV alkalinisation. CQ (in its neutral form) diffuses across the membranes separating the extracellular medium from the DV, but on reaching the acidic interior of the DV becomes protonated (mostly diprotonated) and, hence, less membrane-permeant. The efflux of CQ, in its protonated form and/or in symport with H+, from the DV of CQR parasites, [via the mutated PfCRT (K76T) protein], constitutes a new (verapamil-sensitive) H+ leak which is responsible for the CQ-associated increase in the rate of alkalinisation following inhibition of the V-type H+-ATPase. The circled question mark indicates the possibility that the flux of H+ via mutated PfCRT underlies the higher rate of alkalinisation (following pump inhibition) seen in CQR parasites, as well as the possibility that the efflux of CQ via mutated PfCRT is a H+-coupled (and therefore secondary active transport) process. RBC, red blood cell.
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) and presence (
) of the H+ ionophore CCCP (100 nM). CQ and CCCP were added to saponin-isolated mature trophozoites 1 minute prior to the addition of the H+ pump inhibitor concanamycin A (100 nM). Data are averaged from three separate experiments for D10 and from four separate experiments for 7G8, and are shown as mean ± s.e.m. Where not shown, error bars fall within the symbol. (B) CQ-concentration-dependence of the initial rate of alkalinisation of the DV in CQR 7G8 parasites (estimated from the initial slope of the fluorescence traces) following inhibition of the H+ pump by concanamycin A (100 nM). CQ was added to saponin-isolated mature trophozoites 1 minute before the addition of concanamycin A. The fluorescence ratio was normalised between experiments by dividing by the maximum fluorescence ratio. The initial rate of alkalinisation in the absence of CQ was subtracted from the initial rates in the presence of CQ. The data (± s.e.m.) are averaged from four independent experiments; the line was drawn using a rectangular hyperbola, fitted to the data [y=ax/(b+x), where y is the initial rate of alkalinisation, x is the CQ concentration, a=0.06 second–1 and b=23 µM].