title: A CLAG3 mutation in an amphipathic transmembrane domain alters malaria parasite nutrient channels and confers leupeptin resistance.
abstract: 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 α-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.
authors: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA.
authors: Paresh
authors: P
authors: Sharma
authors: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA.
authors: Kempaiah
authors: K
authors: Rayavara
authors: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA.
authors: Daisuke
authors: D
authors: Ito
authors: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA.
authors: Katherine
authors: K
authors: Basore
authors: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA sdesai@niaid.nih.gov.
authors: Sanjay A
authors: SA
authors: Desai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000502:physiology*; D003198:Computer Simulation; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004351:Drug Resistance / Q000235:genetics / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology; D018503:Genome, Protozoan; D007976:Leupeptins / Q000494:pharmacology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D010963:Plasmodium falciparum / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D015800:Protozoan Proteins; C032854:leupeptin