The Plasmodial Surface Anion Channel And Malaria Parasite Nutrient Acquisition

疟原虫表面阴离子通道与疟原虫营养获取

• 批准号: 7964438
• 负责人: SANJAY A DESAI
• 金额: $ 67.02万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7964438
• 关键词: Affinity; Alleles; Anions; Antibodies; Baculoviruses; Basic Science; Behavior; Biochemical; Biological; Biological Assay; Cell membrane; Cells; Cellular biology; Chemistry; Collection; Complex; Cytolysis; Dependence; Erythrocytes; Exhibits; Family; Furosemide; Gene Family; Genes; Genetic; Genetic Crosses; Goals; Growth; Human; Immune Sera; Immunoprecipitation; Informatics; Inherited; Insecta; Integral Membrane Protein; Ion Channel; Ion Transport; Isotonic Exercise; Laboratories; Lipid Biochemistry; Liposomes; Malaria; Measurement; Mediating; Meiosis; Membrane; Membrane Proteins; Modeling; Molecular; Molecular and Cellular Biology; Nutrient; Parasites; Parents; Pathway interactions; Peptide Hydrolases; Permeability; Physiology; Plasma; Plasmodium falciparum; Primates; Property; Proteins; Role; Route; Signal Transduction; Sorbitol; Specificity; Structure-Activity Relationship; Surface; System; Temperature; Therapeutic; Transmembrane Domain; Transmembrane Transport; Work; base; gene cloning; high throughput screening; inhibitor/antagonist; interest; novel; patch clamp; pressure; protein purification; reconstitution; small molecule libraries; solute; vector

The Apicomplexan Molecular Physiology Section continued its studies of the plasmodial surface anion channel (PSAC) and made two significant contributions. First, we identified stable differences in furosemide efficacy against PSAC activity induced by two common laboratory isolates of P. falciparum, HB3 and 3D7A. This difference was apparent in both single PSAC patch-clamp recordings and in sorbitol-mediated osmotic lysis measurements, confirming that Cl- and sorbitol are transported by a single channel type. HB3 and 3D7A are the parents of a previously executed genetic cross, permitting examination of how the difference in furosemide affinity is inherited by the progeny of this cross. We examined 19 progeny and found complex, non-Mendelian inheritance with some cloned progeny exhibiting furosemide affinities outside the range of parental values. Isolates generated by selfing of the 3D7A clone also exhibited altered furosemide affinities, implicating changes in one or more alleles during meiosis or passage through a primate host. PSAC may be encoded by multiple parasite genes (e.g. a multi-gene family or multiple genes that encode distinct channel subunits) or a single polymorphic gene under strong selective pressure. Second, we have studied how PSAC recognizes and transports a diverse collection of nutrients and inorganic solutes. Despite this broad range of substrates, this channel nevertheless excludes Na+, a feature required to maintain erythrocyte osmotic stability in plasma. Another surprising property of PSAC is its small single-channel conductance (<3 pS in isotonic Cl-) in spite of broad permeability to bulky solutes. While exploring the mechanisms underlying these properties, we identified interactions between permeating solutes and PSAC inhibitors that suggest the channel has more than one route for passage of solutes. 22 structurally diverse solutes were studied with a quantitative osmotic lysis assay. These studies determined that two clearly separated groups of solutes based on their effects on inhibitor affinity, the temperature dependence of these effects and behavior in permeant solute mixtures. The clear separation into two discrete groups suggests two distinct mechanisms of transport through this channel. In contrast to most other broad-permeability channels, selectivity in PSAC appears to be complex and cannot be adequately explained by simple models that invoke sieving through rigid, non-interacting pores. In addition to our studies on PSAC, we have identified a family of three novel integral membrane proteins colocalizing on the inner membrane complex immediately beneath the merozoite plasma membrane. These proteins are interesting because very few polytopic membrane proteins are known to be present in merozoites. Each of these proteins has six predicted transmembrane domains and is conserved in diverse apicomplexan parasites. Immunoprecipitation studies using specific antibodies reveal that these proteins assemble into a heteromeric complex. Each protein was also expressed on insect cells using the baculovirus vector system with a truncated SUMO tag that facilitates maximal expression and protein purification while permitting cleavage with SUMO protease to release unmodified parasite protein. The expressed proteins were successfully reconstituted into artificial liposomes, but were not recognized by human immune sera. Because all three genes are highly conserved in apicomplexan parasites, the complex formed by their encoded proteins likely serves an essential role for invasive merozoites; possibilities include signal transduction or transmembrane transport.

顶复合体分子生理组继续对plasmodial surface阴离子通道（PSAC）进行研究，并做出了两项重要贡献。