Fig. 5: Peptide mimics are substrates of PfCRT in situ.

a Left: schematic showing the H⁺-efflux assay. If a protonated solute is a substrate of PfCRT it will cause a H⁺ leak when effluxed from the DV. Inhibition of the DV’s H⁺-ATPase by concanamycin A enables this H⁺ leak to be detected (with a pH-sensitive probe) as an increase in the rate of DV alkalinisation. Right: several peptide mimics (10 μM) increase the rate of DV alkalinisation in C2^GC03, C6^7G8 and C4^Dd2 parasites. CQ (the positive control) causes the rate of DV alkalinisation to increase in the C6^7G8 and C4^Dd2 lines and to decrease slightly in C2^GC03 parasites. This is consistent with previous applications of the H⁺-efflux assay^36,37 and with the abilities of PfCRT^Ecu1110, PfCRT^7G8 and PfCRT^Dd2 (but not PfCRT^3D7) to transport CQ when tested under normal conditions in the oocyte system^15,16. Unless labelled ns, P < 0.05 relative to the absence of a test solute. b Left: schematic showing the inhibition of PfCRT by verapamil (VP) or the quinine dimer Q₂C in the H⁺-efflux assay. Right: the increase in the rate of DV alkalinisation caused by saquinavir (SQV; 10 μM) and Ac-YF-5-NH₂ (10 μM) is inhibited by VP (50 μM) and Q₂C (1 μM). The statistical analyses were performed relative to the SQV or Ac-YF-5-NH₂ controls. c SQV (left) and Ac-YF-5-NH₂ (right) increase the rate of DV alkalinisation in a concentration-dependent manner. d Concentration-dependence of the trans-stimulation of [³H]CQ transport via PfCRT by SQV (left) and Ac-YF-5-NH₂ (right) in Xenopus oocytes. The non-expressing oocyte data overlays the data obtained with oocytes expressing PfNT1. The data are the mean of n = 5 independent experiments (each yielding similar results and overlaid as individual data points in a and b), and the error is the SEM. Where not visible, the error bars fall within the symbols. The asterisks denote a significant difference from the relevant C4^Dd2 (red asterisks), C6^7G8 (orange asterisks) or C2^GC03 (blue asterisks) control; *P < 0.05, **P < 0.01, ***P < 0.001, ns: non-significant (one-way ANOVA). The source datasets are provided as a Source Data file.