Osmotic lysis measurements

The increased permeability of infected erythrocytes to organic solutes and block by strain-specific inhibitors was quantified using a kinetic assay that tracks 700 nm light transmittance through a suspension of cells [45]. Osmotic lysis resulting from uptake of sorbitol, a sugar alcohol with high PSAC permeability, produces increased light transmittance. Synchronized parasite cultures were enriched at the trophozoite stage with the Percoll-sorbitol method, washed, and resuspended at 37 ^oC in lysis buffer (280 mM sorbitol, 20 mM Na-HEPES, 0.1 mg/ml bovine serum albumin, pH 7.4) to initiate sorbitol uptake and cell swelling that leads to osmotic lysis. Iso-osmotic replacement of sorbitol with either 280 mM proline or 145 mM PhTMA⁺Cl^- was used to measure permeabilities of these solutes; these solutes are representative of the broad range of solutes with PSAC permeability [62]. Inhibitors, where present, were added from DMSO stock solutions without preincubation; control experiments excluded DMSO effects on uptake measurements. 700 nm light transmittance was then continuously tracked in a DU640 or DU800 spectrophotometer (Beckman Coulter). Inhibitor dose-responses and reductions in permeability in transfectant parasites were then calculated from the time required to reach a fractional lysis threshold, based on a conservative two-compartment model of infected cell osmotic lysis in permeant solutes [46]. This method produces estimates of permeability coefficients and inhibitor affinities that quantitatively match those obtained with patch-clamp methods [70].

ISPA-1 was identified as a strain-specific PSAC inhibitor in prior high-throughput screens using four clonal laboratory strains of P. falciparum (Dd2, HB3, 3D7, and Indo 1; [12]). Transport surveys identified this compound as suitable for genetic mapping in the GB4 x 7G8 cross. Dose response studies revealed that ISPA-1 block was adequately fitted by a single Langmuir isotherm with normalized sorbitol permeability, P, given by P = a/ [1 + (x/b)] where a and b are constants. A 10 μM concentration of ISPA-1 produced the greatest difference in parental phenotypes and was used to examine block in progeny clones. Dose responses for block by ISPA-28, a strain-specific inhibitor with potent and specific block of channels linked to the Dd2 clag3.1 gene [12], required fitting to a sum of two Langmuir isotherms, as reported previously [37].