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. 2007 Jan 16;104(3):1063-8.
doi: 10.1073/pnas.0610353104. Epub 2007 Jan 9.

A blasticidin S-resistant Plasmodium falciparum mutant with a defective plasmodial surface anion channel

David A Hill  1 , Ajay D Pillai, Fatima Nawaz, Karen Hayton, Lanxuan Doan, Godfrey Lisk, Sanjay A Desai
Affiliations

A blasticidin S-resistant Plasmodium falciparum mutant with a defective plasmodial surface anion channel

David A Hill et al. Proc Natl Acad Sci U S A. .

Abstract

Erythrocytes infected with malaria parasites exhibit marked increases in permeability to organic and inorganic solutes. The plasmodial surface anion channel (PSAC), an unusual voltage-dependent ion channel induced on the host membrane after infection, may play a central role in these permeability changes. Here, we identified a functional PSAC mutant through in vitro selection with blasticidin S. Resistance to blasticidin S was generated during culture and correlated with significant reductions in permeability to multiple solutes, consistent with uptake via a common pathway. Single channel recordings revealed marked changes in PSAC gating with the addition of a subconductance state not present in wild-type channels. The channel's selectivity profile and pharmacology also were significantly altered. Eventual loss of the mutant phenotype upon removal of selective pressure and slower growth of mutant parasites suggest that PSAC serves an important role in intracellular parasite survival. These findings provide solid evidence for the uptake of diverse solutes via PSAC and implicate one or more parasite genes in expression of this channel.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The blasticidin S-resistant mutant, FCB-br1, exhibits reduced permeability to diverse solutes. (A) Osmotic lysis kinetics at 37°C in isotonic solutions of sorbitol, PhTMA-Cl, or proline, as indicated. In each graph, upper and lower traces represent lysis kinetics for FCB and FCB-br1, respectively. Lysis of FCB-br1 is slower in each solute, indicating reduced permeability across the host erythrocyte membrane. (B) Ratios of permeability coefficients (PFCB/PFCB-br1) for the solutes in A, shown as the mean ± SEM of four to five measurements. Permeability coefficients were calculated as the reciprocal of the time to 50% lysis, as formally justified for passive diffusion via PSAC (23). (C) Immunoblot showing reduced labeling of hemoglobin by sulfosuccinimidyl-6-(biotinamido)hexanoate in FCB-br1 parasites (lane 2) when compared with FCB parasites (lane 1), as detected with a monoclonal antibody against biotin. The blot was stripped and reprobed with an anti-hemoglobin antibody to confirm equal protein loading. Because the extent of hemoglobin labeling correlates with reagent permeation via PSAC (4), the blot reflects reduced reagent permeability in FCB-br1.
Fig. 2.
Fig. 2.
Electrophysiological differences between FCB and FCB-br1. (A) Whole-cell voltage-clamp recordings from erythrocytes infected with FCB or FCB-br1 trophozoites with solution A in both bath and pipette compartments. Each group of traces represents superimposed current responses to 50-ms pulses from a holding potential of 0 mV to values between −100 and +100 mV in 10-mV increments. The FCB-br1 isolate induces markedly smaller Cl− currents. (B) Single channel recordings in solution A at imposed membrane potential (Vm) of −100 mV. Whereas PSAC activity on FCB is indistinguishable from that on other wild-type isolates, activity on FCB-br1 is visibly altered. Black dashes on the sides of each trace represent the closed channel level; the subconductance level seen on FCB-br1 is demarcated with a red line behind each of those traces. (Scale bars: A, 25 ms and 1.0 nA; B, 6.7 ms and 0.83 pA.) (C) All points histograms calculated from single channel recordings as in B for FCB and FCB-br1 isolates (black and red traces, respectively). Each histogram appears as a smooth curve because of narrow bin widths (0.02 pA) and calculation from ≈2 × 106 samples (20 s of single channel recording). The mean closed (c), open (o), and subconductance (s) levels are marked with one-letter abbreviations.
Fig. 3.
Fig. 3.
Markedly reduced sensitivity to furosemide of PSAC expressed on FCB-br1. (A) (Upper) Osmotic lysis kinetics in proline at 20°C in the presence of 0, 10, 25, and 200 μM furosemide (top to bottom traces in each graph, respectively). Proline uptake by FCB-br1 is less well inhibited by furosemide. (Lower) The corresponding dose responses for inhibition. Symbols represent the mean ± SEM lysis rates normalized to 1.0 without furosemide, calculated as described in ref. from up to five measurements at each concentration. Solid lines represent a least-squares best fit to y = K0.5/(K0.5 + x) with K0.5 estimates of 0.8 and 32 μM for FCB and FCB-br1, respectively. (B) Single PSAC recordings in solution A with 10 μM furosemide in bath and pipette. Vm = −100 mV. (Scale bars: 100 ms and 2 pA.) Closed channel currents are indicated by dashes to the sides of each trace. Furosemide significantly reduces channel openings (downward deflections from the closed channel level) for PSAC on the FCB isolate but has a negligible effect on FCB-br1 channels when compared with their activities without inhibitor (Fig. 2).
Fig. 4.
Fig. 4.
Loss of resistance to blasticidin S is linked to the reappearance of wild-type PSAC activity. (A) Osmotic lysis kinetics in sorbitol for FCB (black trace), FCB-br1 (red), FCB-br1-rev (green), and FCB-br2 (blue). Wild-type sorbitol permeability is restored in the blasticidin S-sensitive revertant; subsequent selection of blasticidin S resistance yields reduced sorbitol permeability, similar to that seen with FCB-br1. (B) Correlation between in vitro growth inhibition by blasticidin S (red bar, normalized to 1 for complete microscopic clearance of parasites after 4 days in culture with blasticidin S), whole-cell chord conductance in solution A (blue bar, mean ± SEM of n = 10–29 cells for each isolate), and apparent sorbitol permeability coefficient (green bars, mean ± SEM of n = 3–6 trials for each isolate, calculated as the reciprocal of the lysis halftime). (C) Single PSAC recordings with the same conditions as in Fig. 2B. (Scale bars: 50 ms and 2 pA.) Whereas PSAC on FCB-br1-rev exhibits wild-type gating, FCB-br2 channel gating resembles that of FCB-br1.
Fig. 5.
Fig. 5.
In vitro growth rates for FCB (circles) and FCB-br1 (triangles) with and without blasticidin S (open and filled symbols, respectively) quantified by using SYBR Green I-based fluorescent detection of parasite DNA in arbitrary units. Experiments evaluating growth rates by microscopic determination of parasitemias produced similar results (data not shown).
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