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: 7592254
• 负责人: SANJAY A DESAI
• 金额: $ 75.53万
• 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/7592254
• 关键词: Affect; Affinity; Alanine; Ammonium; Anions; Antimalarials; Basic Science; Biochemical; Biological; Biological Assay; Biophysics; Cells; Cellular biology; Cloning; Collection; Dependence; Drug resistance; Electrophysiology (science); Erythrocytes; Exhibits; Genes; Genetic Materials; Goals; Growth; Human; In Vitro; Infection; Informatics; Ion Channel; Ion Transport; Isoleucine; Lead; Libraries; Lipid Biochemistry; Malaria; Mediating; Membrane; Membrane Proteins; Modeling; Molecular; Molecular Biology; Numbers; Nutrient; Parasites; Pathway interactions; Permeability; Pharmacology; Phenotype; Physiology; Predisposition; Property; Proteins; Range; Resistance; Role; Route; Sorbitol; Specificity; Structure-Activity Relationship; Surface; Temperature; Therapeutic; Thinking; Toxin; Work; blasticidin S; cost; drug development; fitness; gene cloning; high throughput screening; inhibitor/antagonist; killings; mutant; novel strategies; resistance mechanism; small molecule; solute; uptake

The Apicomplexan Molecular Physiology Section continued its studies of the plasmodial surface anion channel (PSAC) and made two significant contributions. First, we identified and characterized the first PSAC mutant (PNAS 104:1063-1068, 2007). This mutant was generated by in vitro selection with blasticidin S, a toxin that reaches its intracellular target via permeation through PSAC. Because blasticidin S resistance correlated with reduced permeability to multiple solutes, we predicted there may be marked changes in PSAC. Single-channel and whole-cell electrophysiology confirmed this prediction by revealing altered channel gating, selectivity, pharmacology, and functional copy number/infected cell. Mutant parasites cultured without blasticidin S reverted to the wild-type channel phenotype and exhibited restored susceptibility to killing by blasticidin S. These findings 1) confirm that PSAC is the primary mechanism of organic solute uptake after infection because changes in PSAC affected the permeability of each solute, 2) implicate parasite genes in the expression of PSAC because human erythrocytes lack heritable genetic material, 3) provide a new approach to cloning PSACs gene, 4) reveal a new drug resistance mechanism in malaria parasites, and 5) suggest that PSAC serves an essential role for the intracellular parasite because a fitness cost was associated with blasticidin S resistance. Second, we identified solute-inhibitor interactions within PSACs pore (Mol. Pharmacol. 71:1241-50, 2007). In this study, we found that phenyltrimethyl ammonium and isoleucine transport through PSAC were less effectively inhibited by known PSAC antagonists than the uptake of sorbitol and alanine. This observation was unexpected because all four solutes were thought to share a single transport mechanism. We excluded uptake via unrelated channels because specific PSAC inhibitors also exhibited solute-dependent affinities. Mixtures of permeating solutes, whole-cell electrophysiology, and temperature-dependence studies suggested that a single ion channel with two separate routes for permeating solutes is the most conservative explanation for our findings. Such a model may permit fine-tuning of PSACs unusual selectivity and allow the parasite to acquire a diverse collection of nutrients. These findings also suggest that PSAC antagonists must effectively block both routes to be suitable lead compounds for antimalarial drug development.

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，我们鉴定并表征了第一个PSAC突变体（PNAS 104：1063-1068，2007）。 该突变体通过用杀稻瘟菌素S（一种通过渗透通过PSAC到达其细胞内靶标的毒素）进行体外选择而产生。 由于杀稻瘟菌素S抗性与对多种溶质的渗透性降低相关，因此我们预测PSAC可能存在显著变化。 单通道和全细胞电生理学通过揭示改变的通道门控、选择性、药理学和功能拷贝数/感染细胞证实了这一预测。 在没有杀稻瘟菌素S的情况下培养的突变寄生虫恢复到野生型通道表型，并表现出恢复的对杀稻瘟菌素S杀伤的敏感性。 这些发现1）证实了PSAC是感染后有机溶质摄取的主要机制，因为PSAC的变化影响了每种溶质的渗透性，2）由于人类红细胞缺乏可遗传的遗传物质，因此PSAC的表达涉及寄生虫基因，3）提供了克隆PSAC基因的新方法，4）揭示了疟疾寄生虫中新的耐药机制，和5）表明PSAC对于胞内寄生物起重要作用，因为适合度成本与杀稻瘟菌素S抗性相关。 其次，我们确定了PSAC孔内的溶质-抑制剂相互作用（Mol. Pharmacol. 71：1241-50，2007）。 在这项研究中，我们发现，苯三甲基铵和异亮氨酸运输通过PSAC被有效抑制已知的PSAC拮抗剂比山梨糖醇和丙氨酸的摄取。 这一观察结果是出乎意料的，因为所有四种溶质被认为共享一个单一的运输机制。 我们排除了通过不相关通道的摄取，因为特异性PSAC抑制剂也表现出溶质依赖性亲和力。 渗透溶质的混合物，全细胞电生理学和温度依赖性研究表明，一个单一的离子通道与两个独立的路线渗透溶质是我们的研究结果最保守的解释。 这样的模型可以允许PSAC不寻常的选择性微调，并允许寄生虫获得不同的营养素集合。 这些发现还表明，PSAC拮抗剂必须有效地阻断这两种途径，才能成为抗疟药物开发的合适先导化合物。