Erythrocytes infected with malaria parasites have increased permeability to ions and nutrients, as mediated by the plasmodial surface anion channel (PSAC) and recently linked to parasite clag3 genes. Although the encoded protein is integral to the host membrane, its precise contribution to solute transport remains unclear because it lacks conventional transmembrane domains and does not have homology to ion channel proteins in other organisms. Here, we identified a probable CLAG3 transmembrane domain adjacent to a variant extracellular motif. Helical-wheel analysis revealed strict segregation of polar and hydrophobic residues to opposite faces of a predicted ␣-helical transmembrane domain, suggesting that the domain lines a water-filled pore. A single CLAG3 mutation (A1210T) in a leupeptin-resistant PSAC mutant falls within this transmembrane domain and may affect pore structure. Allelic-exchange transfection and site-directed mutagenesis revealed that this mutation alters solute selectivity in the channel. The A1210T mutation also reduces the blocking affinity of PSAC inhibitors that bind on opposite channel faces, consistent with global changes in channel structure. Transfected parasites carrying this mutation survived a leupeptin challenge significantly better than a transfection control did. Thus, the A1210T mutation contributes directly to both altered PSAC activity and leupeptin resistance. These findings reveal the molecular basis of a novel antimalarial drug resistance mechanism, provide a framework for determining the channel's composition and structure, and should guide the development of therapies targeting the PSAC.T he human malaria parasite Plasmodium falciparum remodels its host erythrocyte by exporting many proteins, generating membranous structures in the host cytosol, and increasing erythrocyte permeability to many solutes. Studies by multiple groups have determined that anions, sugars, purines, organic cations, and some vitamins have increased permeability after infection (1-4). The increase in permeability is primarily mediated by a parasitederived ion and nutrient channel known as the plasmodial surface anion channel (PSAC) (5). Importantly, both PSAC single-channel properties and the relative increases in solute permeabilities are conserved in divergent malaria parasites (6). Because Babesia parasites do not induce PSAC-like activity in erythrocytes that they invade (7), this channel is thought to be restricted to the genus Plasmodium.The clag multigene family, also conserved in and restricted to malaria parasites (8), has recently been linked to PSAC activity (9-11). Two paralogs on parasite chromosome 3, known as clag3.1 and clag3.2, appear to play a critical role, as identified by genetic mapping experiments with ISPA-28, an isolate-specific PSAC antagonist that blocks channels on the Dd2 laboratory clone but is ineffective against channels from other lines. These genes undergo epigenetic switching to encode a single protein that is initially packaged in specialized organelles known as rh...