In 2012, the Apicomplexan Molecular Physiology Section continued studies into the molecular basis of increased erythrocyte permeability after infection with malaria parasites. We previously identified the plasmodial surface anion channel (PSAC), an unusual ion channel only on the erythrocyte membrane of infected cells. Other groups confirmed our finding of increased permeability, but proposed multiple distinct ion channels on the host membrane. Therefore, important unknowns were 1) the number of distinct ion channels, and 2) the molecular basis of these channels. We resolved these unknowns recently by identifying parasite clag3 genes as determinants of PSAC activity (Cell 145:665-677, 2011). The encoded protein localizes to the host erythrocyte membrane;functional studies of transfectant parasites indicated that PSAC is the principal ion channel for small organic solutes at the host membrane. Gene identification has raised important new questions that we have taken up in 2012. Although clag3 gene expression is critical for PSAC activity, it is unclear whether the encoded protein contributes directly to formation of the channel pore. We have now obtained early insights into this question with studies of voltage-dependent inactivation in PSAC. We determined that ion flow through PSAC decreases upon continuous application of negative membrane potentials, a phenomenon known as inactivation. In contrast to inactivation in mammalian ion channels, this decrease required washout of the erythrocyte cytosol and could only be detected with the whole-cell patch-clamp configuration. Addition of intracellular protease in whole-cell patch-clamp prevented inactivation and implicated a negatively charged cytoplasmic component of the channel. Consistent with these predictions, computational analysis of the CLAG3 protein reveals a marked enrichment of negatively charged residues at the C-terminal cytoplasmic tail. These findings suggest that the CLAG3 protein contributes directly to the functioning ion channel and that its cytoplasmic tail is tethered to one or more soluble proteins in the erythrocyte cytosol. DNA transfection experiments to test this model are underway. In 2012, we also developed a new fluorescence-based method for detection of parasite growth in microwell plates. The method uses the C-SNARF pH sensitive dye to measure the reduction in extracellular pH associated with malaria parasite growth. Because this dye is nontoxic to parasite cultures, we determined that C-SNARF can be used to detect parasite growth without partial transfer of cultures to assay plates;this is important because transfer of cultures adds significantly to the cost of detection and consumes precious small-volume cultures. Our optimized assay produces robust and high-throughput detection of parasite growth. This new method will be useful for limiting dilution cloning of malaria parasite cultures, as frequently employed in clinical studies of isolates from patient blood samples and in basic research studies using molecular and genetic techniques.
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