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.

顶复门分子生理学组继续研究疟原虫表面阴离子通道（PSAC），并作出了两项重大贡献。 首先，我们确定了呋塞米对两种常见的恶性疟原虫实验室分离株HB 3和3D 7A诱导的PSAC活性的疗效的稳定差异。这种差异是明显的，在这两个单一的PSAC膜片钳记录和山梨醇介导的渗透溶解测量，确认Cl-和山梨醇通过一个单一的通道类型运输。HB 3和3D 7A是先前执行的遗传杂交的亲本，允许检查呋塞米亲和力的差异如何被该杂交的后代遗传。我们检查了19个后代，发现复杂的，非孟德尔遗传与一些克隆后代表现出呋塞米亲和力的范围外的亲本值。通过3D 7A克隆自交产生的分离物也表现出改变的呋塞米亲和力，暗示减数分裂或通过灵长类宿主期间一个或多个等位基因的变化。PSAC可以由多个寄生虫基因（例如多基因家族或编码不同通道亚基的多个基因）或在强选择压力下的单个多态性基因编码。 其次，我们研究了PSAC如何识别和运输多种营养物质和无机溶质。 尽管底物范围很广，但该通道仍然排除了Na+，这是维持血浆中红细胞渗透稳定性所需的特征。PSAC的另一个令人惊讶的性质是其小的单通道电导（在等渗Cl-中<3 pS），尽管对大体积溶质具有宽的渗透性。在探索这些特性的机制时，我们确定了渗透溶质和PSAC抑制剂之间的相互作用，这表明通道具有不止一种溶质通过的途径。 用定量渗透溶解试验研究了22种结构不同的溶质。 这些研究确定了两组明显分离的溶质，基于它们对抑制剂亲和力的影响，这些影响的温度依赖性和渗透性溶质混合物中的行为。明显分离为两个离散的组表明通过该通道的两种不同的运输机制。与大多数其他宽渗透性通道相反，PSAC中的选择性似乎是复杂的，并且不能通过简单的模型来充分解释，该模型通过刚性的、非相互作用的孔来进行筛选。 除了我们对PSAC的研究，我们已经确定了一个家庭的三个新的整合膜蛋白共定位于内膜复合物紧下面的裂殖子质膜。 这些蛋白是有趣的，因为很少有多位膜蛋白是已知的裂殖子中存在。 这些蛋白质中的每一个都具有六个预测的跨膜结构域，并且在不同的顶复门寄生虫中是保守的。使用特异性抗体的免疫沉淀研究表明，这些蛋白质组装成异聚复合物。还使用具有截短的SUMO标签的杆状病毒载体系统在昆虫细胞上表达每种蛋白，所述截短的SUMO标签促进最大表达和蛋白纯化，同时允许用SUMO蛋白酶切割以释放未修饰的寄生虫蛋白。表达的蛋白成功地重组到人工脂质体中，但不被人免疫血清识别。由于这三个基因在顶复门寄生虫中高度保守，因此它们编码的蛋白质形成的复合物可能对侵入性裂殖子起重要作用;可能性包括信号转导或跨膜转运。