Plasmodial Anion Channel/Malaria Parasite Nutrient

Apicomplexan分子生理学单位对感染疟疾寄生虫的人类红血球的各种膜上离子和营养物质的运输进行基础研究。这项工作包括分子生物学和信息学，蛋白质和脂肪生物化学，膜蛋白的免疫荧光定位，各种转运分析，生物物理学，化合物文库的高通量筛选，以及小分子抑制剂的构效关系研究。 我们之前在感染恶性疟原虫的人红细胞上发现了一种不寻常的离子通道，恶性疟原虫是最致命的疟疾形式。这种通道，即原生物质表面阴离子通道(PSAC)，存在于1000个拷贝/细胞中，具有不同寻常的门控特性，可以渗透到一系列已知的寄生虫生长所需的阴离子和营养物质中。我们认为PSAC介导了营养获得的连续扩散途径的第一步。目前实验室中的项目包括：1)检测PSAC门控和选择性特性的功能研究；2)高通量筛选以寻找具有治疗潜力的高亲和力、高特异性的PSAC拮抗剂；3)旨在克隆编码PSAC和其他转运体的基因(S)的生化和分子生物学研究；以及4)这些转运体的异源表达。我们这些项目的总体目标是探索PSAC如何实现其不同寻常的功能特性，了解寄生虫的细胞生物学和生理学，并开发控制疟疾的新策略。 在过去的一个财政年度，实验室在这一重要领域做出了几项贡献。最重要的是，我们研究了PSAC是一种被运输到宿主红细胞膜的寄生虫编码蛋白还是一种修饰的宿主膜蛋白的基本争论。在这些研究中，我们首次发现，当红细胞与地理上不同的寄生虫分离株培养时，PSAC门控行为的多态，表明PSAC是由寄生虫编码的。第二个成就是将丹曲林鉴定为一种不寻常的PSAC拮抗剂，为其体外寄生虫生长抑制作用提供了解释。然后，对164个丹曲烯衍生物进行二次筛选，确定了比任何先前已知的PSAC拮抗剂都具有更高特异性和亲和力的化合物。最后，我们在有无添加拮抗剂的情况下探索通道门控，以确定PSAC只有一个开放状态和至少三个关闭状态。我们的研究为这一不同寻常的通道的结构和功能提供了新的见解。

The Apicomplexan Molecular Physiology Unit conducts basic research on the transport of ions and nutrients across various membranes of human red blood cells infected with malaria parasites. This work incorporates molecular biology and informatics, protein and lipid biochemistry, immunofluorescent localization of membrane proteins, various transport assays, biophysics, high-throughput screening of compound libraries, and examination of structure-activity relationships for small molecule inhibitors. We previously identified an unusual ion channel on human red blood cells infected with P. falciparum, which causes the deadliest form of malaria. This channel, the plasmodial surface anion channel (PSAC), is present at 1000 copies/cell, has unusual gating properties, and is permeable to a range of anions and nutrients known to be required for parasite growth. We proposed that PSAC mediates the first step in a sequential diffusive pathway of nutrient acquisition. Current projects in the lab include: 1) functional studies to examine PSAC gating and selectivity properties, 2) high-throughput screening to identify high affinity, high specificity PSAC antagonists with therapeutic potential, 3) biochemical and molecular biological studies aimed at cloning the gene(s) encoding PSAC and other transporters, and 4) heterologous expression of these transporters. Our overall goal with these projects is to probe how PSAC achieves its unusual functional properties, to understand the parasite's cell biology and physiology, and to develop new strategies for the control of malaria. In the past fiscal year, the lab made several contributions to this important field. Most importantly, we examined a fundamental debate on whether PSAC is a parasite-encoded protein trafficked to the host RBC membrane or a modified host membrane protein. In these studies, we identified, for the first time, polymorphisms in PSAC gating behavior when red cells are cultured with geographically distinct parasite isolates, suggesting that PSAC is parasite-encoded. A second accomplishment is the identification of dantrolene as an unusual PSAC antagonist, providing an explanation for its in vitro parasite growth inhibitory effects. A secondary screen of 164 dantrolene derivatives then identified compounds of higher specificity and affinity than any previously known PSAC antagonists. Finally, we probed channel gating with and without added antagonists to determine that PSAC has only one open and at least three closed states. Our studies have provided new insights into this unusual channel's structure and function.