The Plasmodial Surface Anion Channel And Malaria Parasite Nutrient Acquisition

Apicomplexan分子生理学研究组继续对原虫表面阴离子通道(PSAC)进行研究，并做出了两项重大贡献。 首先，我们鉴定并鉴定了第一个PSAC突变体(PNAS 104：1063-1068,2007)。该突变体是用杀菌素S进行体外筛选而产生的，杀菌素是一种通过PSAC渗透到达其细胞内目标的毒素。由于杀菌素对S的抗性与多种溶质的渗透性降低有关，因此我们预测PSAC可能会发生显著变化。单通道和全细胞电生理学证实了这一预测，揭示了通道门控、选择性、药理学和功能拷贝数/感染细胞的变化。没有杀菌素S培养的突变寄生虫回复到野生型通道表型，并表现出对杀菌素S杀灭的恢复敏感性。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通透性；2)由于人红细胞缺乏可遗传的遗传物质，PSAC基因的表达涉及到寄生虫基因的表达；3)提供了克隆PSACS基因的新途径；4)揭示了疟疾寄生虫的一种新的抗药性机制；5)表明PSAC对细胞内寄生虫起重要作用，因为适应成本与S的抗药性有关。 其次，我们确定了PSACs孔内的溶质-缓蚀剂相互作用(Mol.药水。71：1241-50,2007)。在这项研究中，我们发现已知的PSAC拮抗剂对苯三甲基铵和异亮氨酸通过PSAC的转运的抑制作用不如山梨醇和丙氨酸的摄取有效。这一观察结果是出乎意料的，因为所有四种溶质都被认为共享一种传输机制。我们排除了通过无关渠道的摄取，因为特定的PSAC抑制剂也表现出溶质依赖的亲和力。渗透溶质、全细胞电生理学和温度依赖研究的混合表明，具有两条不同渗透途径的单一离子通道是对我们的发现最保守的解释。这样的模型可能允许微调PSACs的异常选择性，并允许寄生虫获得不同的营养集合。这些发现还表明，PSAC拮抗剂必须有效地阻断这两条途径，才能成为适合抗疟疾药物开发的先导化合物。

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.