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

基本信息

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
项目类别：
财政年份：
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

项目摘要

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.

Apicomplexan分子生理学部分继续研究了质体表面阴离子通道（PSAC），并做出了两个重要的贡献。首先，我们确定了由恶性疟原虫，HB3和3D7A的两个常见实验室分离株引起的速尿疗法效应稳定的差异。这种差异在单个PSAC斑块钳记录和山梨糖介导的渗透裂解测量中都显而易见，这证实了Cl-和山梨糖醇通过单个通道类型运输。 HB3和3D7A是先前执行的遗传十字架的父母，允许研究该十字架的后代如何遗传速尿相关性的差异。我们检查了19个后代，发现了复杂的，非孟德尔的遗传，一些克隆的后代在父母价值的范围之外表现出速尿相关性。 3D7A克隆产生的分离株也表现出改变的速尿相关性，暗示了减数分裂或通过灵长类动物宿主的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，一个多基因家族或编码不同通道亚基的多个基因）或在强选择性压力下单个多态基因编码。其次，我们研究了PSAC如何识别和运输各种营养和无机溶质。尽管存在广泛的底物，但该通道仍然不包括Na+，这是维持血浆中红细胞渗透稳定性所需的功能。 PSAC的另一个令人惊讶的特性是，尽管对笨重的溶质具有广泛的渗透性，但其单通道电导率很小（等渗Cl-中的<3 ps）。在探索这些特性基础的机制时，我们确定了渗透溶质和PSAC抑制剂之间的相互作用，这些溶质与PSAC抑制剂表明该通道具有多种溶质的途径。用定量的渗透裂解测定法研究了22个结构上多样的溶质。这些研究确定了两个明确分离的溶质基，这些溶质对抑制剂亲和力的影响，这些作用和行为的温度依赖性和渗透性溶质混合物的温度依赖性。将两个离散组的明确分离提出了通过该通道的两种不同的运输机制。与大多数其他透明度通道相反，PSAC中的选择性似乎很复杂，无法通过简单的模型来充分解释，这些模型通过刚性的，非相互作用的毛孔唤起筛分。除了对PSAC的研究外，我们还确定了一个三个新型整合膜蛋白的家族，在梅洛唑岩质膜下方的内膜复合物上共定位。这些蛋白很有趣，因为已知在梅罗寄生虫中存在的多重膜蛋白很少。这些蛋白质中的每一个都有六个预测的跨膜结构域，并在不同的丙糖寄生虫中保守。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类络合物。每种蛋白质还使用带有截短的SUMO标签的杆状病毒载体系统在昆虫细胞上表达，该标签促进了最大表达和蛋白质纯化，同时允许使用Sumo蛋白酶裂解以释放未修饰的寄生虫蛋白质。表达的蛋白质成功地重构为人造脂质体，但没有被人类免疫血清认可。由于所有三个基因在Apicomplexan寄生虫中都高度保守，因此由其编码蛋白形成的复合物可能对浸润性梅罗寄生虫起着至关重要的作用。可能性包括信号转导或跨膜传输。