Figure 1.

Expression of PfCRT in X. laevis oocytes. A, Western blot analysis of total lysates from oocytes injected with water, PfCRT^HB3 cRNA, and PfCRT^Dd2 cRNA, using a polyclonal guinea pig antiserum specific to PfCRT and a polyclonal rabbit antiserum specific to α-tubulin. A size standard is indicated in kilodaltons. The hybridization signals were subsequently quantified, yielding the expression levels of PfCRT^HB3 and PfCRT^Dd2 relative to the internal standard α-tubulin. The means ± S.E. (error bars) from the number of independent biological replicates N_BR = 4 independent Western blot analyses are shown. B, confocal fluorescence images of fixed PfCRT^Dd2 and PfCRT^Dd2-expressing oocytes and water-injected control oocytes. Left, fluorescence image of InsP₃R, using a specific rabbit antisera and an Alexa Fluor 546 anti-rabbit secondary antibody. Middle, fluorescence image of PfCRT, using a specific guinea pig antiserum and the Alexa Fluor 488 anti-guinea pig secondary antibody. Right, differential interference contrast image. Bar, 250 μm. C, effect of ND10 and ND96 buffer, pH 5.5, on PfCRT^Dd2-mediated chloroquine uptake in the presence and absence of verapamil (VP; 100 μm). Oocytes were incubated in the respective buffer, pH 7.5, from the moment of cRNA injection. For the flux experiments, chloroquine was added at a final concentration of 50 μm unlabeled chloroquine and 42 nm [³H]chloroquine, and the amount of uptake was determined after 60 min of incubation. The means ± S.E. of N_BR = 4 independent biological determinations are shown. Statistical significance was assessed using the two-tailed t test. D, intracellular ATP levels of water-injected oocytes and oocytes expressing PfCRT^HB3 or PfCRT^Dd2. Oocytes were incubated in ND10 buffer, pH 7.5, after cRNA injection, for 3 days before the intracellular ATP levels were determined. The mean ± S.E. of N_BR = 5 independent biological determinations are shown. The statistical significance was assessed using the Holm–Sidak one-way ANOVA test or a two-tailed t test, where appropriate. n.s., not significant.