Figure 4. RhopH complex abundance correlates with PSAC activity.

(A) Osmotic lysis kinetics resulting from sorbitol uptake by wild-type parasites with or without exposure to 4 mM GlcN (red and black traces, respectively). (B–C) Lysis kinetics for transfectant parasites without (black) or with 0.5, 1, 2, or 4 mM GlcN (top to bottom red traces, respectively). Note the extended time scales for osmotic lysis. (D) Normalized sorbitol permeabilities (mean ± S.E.M.) for each parasite at indicated GlcN concentrations. Solid lines represent best fits to y = a/(1 + (x/K_0.5)). (E) Percentage of cells refractory to osmotic lysis (mean ± S.E.M.) at each [GlcN]. Solid lines represent best fits to a two-parameter logistic curve. (F) Whole-cell currents from individual trophozoite-infected erythrocytes, presented as the ensemble responses to membrane voltages (V_m) between −100 and +100 mV in 10 mV increments. 4 mM GlcN specifically abolishes currents on knockdown parasites. Red dashes indicate zero current levels. The schematic on the left shows the whole-cell patch-clamp configuration; the recording pipette measures currents resulting from ion flow through channels on the cell surface; the measured currents correspond to ~4000 channels on infected cells not subjected to GlcN knockdown. (G) Chord conductances (g_Cl) (mean ± S.E.M.) for each parasite without GlcN, calculated from whole-cell currents between V_m of 0 and −100 mV. Statistics were calculated from up to 15 cells each. The value for wild-type cells after 4 mM GlcN treatment is also shown. (H) Whole-cell ensemble currents for transfectants cultivated with 4 mM GlcN, taken from panel (F) but presented at an increased gain. Single traces beneath each ensemble show stochastic transitions that are due to openings of individual channels detected in the whole-cell configuration; V_m, −90 and −70 mV for traces shown at bottom in R2glmS and R3glmS, respectively. These transitions and the reduced ensemble amplitudes indicate a marked reduction in PSAC copy number. Horizontal and vertical scale bars represent 25 ms and 10 pA, respectively, for all traces. The schematic on the left shows the whole-cell patch-clamp configuration and a reduced number of channels. (I) Parasite growth (mean ± S.E.M.) over 72 hr in standard medium or PGIM (black and red bars, respectively) after exposure to 1 mM GlcN. For each parasite, growth was normalized to 100% for matched controls using the same parasite and culture medium without GlcN treatment.

DOI: http://dx.doi.org/10.7554/eLife.23485.007

Figure 4—figure supplement 1. Organic solute permeabilities and cell-attached patch-clamp of wild-type and glmS knockdown parasites.

(A) Permeabilities (P) (mean ± S.E.M.) of the indicated organic solutes in wild-type and transfectant infected cells grown with 1 mM GlcN treatment. PhTMA+, phenyl trimethyl-ammonium cation. Values were determined from osmotic lysis kinetic experiments and normalized to matched cultures without GlcN. Reductions in solute permeabilities were uniform for both transfectant parasites (p>0.9, One-way ANOVA). (B) Cell-attached recordings on erythrocyte membranes from cells infected with indicated parasites without or with 4 mM GlcN treatment. Downward deflections in each trace reflect openings of single channels in the membrane patch. Red dashes indicate closed channel currents. Representative of up to 41 patches in each condition. Imposed membrane protentials, −100 mV; scale bars represent 100 ms horizontal and 2 pA vertical for all traces. The schematic shows cell-attached patch-clamp of an infected erythrocyte; a single PSAC is shown in the membrane patch being recorded.

DOI: http://dx.doi.org/10.7554/eLife.23485.008