In 2014, the Apicomplexan Molecular Physiology Section continued studies into the molecular basis and physiological role of increased erythrocyte permeability after infection with malaria parasites. In one study, we examined how an unusual parasite multigene family contributes to formation of nutrient channels at the host erythrocyte membrane. Previous studies implicated the plasmodial surface anion channel (PSAC) and the clag multigene family in the increased uptake of nutrients and monovalent ions at the erythrocyte surface. An important question in the field at present is how CLAG proteins contribute to channel activity. In one model, these proteins function as enzymes that activate quiescent channels already present on the host cell surface;in another, the CLAG proteins contribute directly to formation of the channel, either in isolation or through interactions with unrelated proteins. This uncertainly is especially important because the clag genes do not have detectable homology to known ion channels in other organisms. In this reporting year, we used proteases to examine the channel's composition. While proteases with distinct specificities all cleaved within an extracellular domain of CLAG3, they produced differing degrees of transport inhibition. Chymotrypsin-induced inhibition depended on parasite genotype, with channels induced by the HB3 parasite affected to a greater extent than those of the Dd2 clone. Inheritance of functional proteolysis in the HB3xDd2 genetic cross, DNA transfection, and gene silencing experiments all pointed to the clag3 genes, providing independent evidence for a role of these genes. Protease protection assays with a Dd2-specific inhibitor and site-directed mutagenesis revealed that a variant L1115F residue on a CLAG3 extracellular loop contributes to inhibitor binding and accounts for differences in functional proteolysis. These findings suggest that surface-exposed CLAG3 contributes directly to channel function;they also provide early structural insights into the PSAC pore. PLoS ONE 9: e93759 (2014). In a second study, we examined the unusual ability of PSAC to identify and distinguish solutes for uptake. This question is important because there are many nutrients and antimalarial drugs that enter infected erythrocytes primarily via PSAC. Despite the broad range of permeant solutes, the channel stringently excludes sodium ions;this exclusion is essential for survival of the intracellular parasite in host plasma. Here, we explored mechanisms for this remarkable solute selectivity and identified guanidinium as an organic cation with high permeability into erythrocytes infected with malaria parasites, but negligible uptake by uninfected cells. Transport characteristics and pharmacology indicated that this uptake is specifically mediated by PSAC. We also examined organic and inorganic cation permeabilities and proposed that cation dehydration is the rate-limiting step in transport through the channel. The high guanidinium permeability of infected cells also allows rapid and stringent synchronization of parasite cultures, as required for molecular and cellular studies of this pathogen. This study provides a framework for nutrient and ion permeation through PSAC. Understanding the structural and molecular basis of permeation is critical to knowing the channel's role in host-parasite interactions and to developing inhibitors that may be future antimalarial drugs. BioMed Research International, in press (2014). In a third study, we examined the increased permeability of infected cells to calcium, an essential divalent cation. We used nondestructive loading of a fluorescent calcium indicator dye (Fluo-8) into human erythrocytes to quantify Ca++ uptake kinetics. Our studies revealed that infection with malaria parasites produces marked increases in erythrocyte Ca++ permeability. Pharmacological studies revealed that this uptake is not mediated by PSAC or by typical mammalian Ca++ channels. Parasite growth inhibition studies revealed a conserved requirement for extracellular Ca++. These findings suggest a novel pathway for Ca++ uptake after infection. Inhibitors of this pathway may be excellent starting points for antimalarial drug development. Malaria J. 13:184 (2014).
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