Plasmodial Anion Channel/Malaria Parasite Nutrient

Apicomplexan分子生理学单位对离子和营养物质穿过感染疟疾寄生虫的人类红细胞的各种膜的运输进行基础研究。这项工作结合了分子生物学和信息学，蛋白质和脂质生物化学，膜蛋白的免疫荧光定位，各种运输试验，生物物理学，化合物库的高通量筛选，以及小分子抑制剂的结构-活性关系的检查。 我们先前在感染恶性疟原虫的人类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。这种通道，疟原虫表面阴离子通道（PSAC），是目前在1000个拷贝/细胞，具有不寻常的门控特性，并渗透到一系列的阴离子和营养素已知所需的寄生虫生长。我们提出，PSAC介导的第一步，在一个连续的扩散途径的养分收购。实验室目前的项目包括：1）功能研究，以检查PSAC门控和选择性特性，2）高通量筛选，以确定具有治疗潜力的高亲和力，高特异性PSAC拮抗剂，3）生物化学和分子生物学研究，旨在克隆编码PSAC和其他转运蛋白的基因，以及4）这些转运蛋白的异源表达。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里，该实验室为这一重要领域做出了多项贡献。最重要的是，我们研究了一个根本的争论，是否PSAC是寄生虫编码的蛋白质贩运到宿主红细胞膜或修饰的宿主膜蛋白。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门控行为的多态性，表明PSAC是寄生虫编码的。第二个成就是丹曲林作为一种不寻常的PSAC拮抗剂的鉴定，为其体外寄生虫生长抑制作用提供了解释。然后，对164种丹曲林衍生物进行二次筛选，鉴定出比任何先前已知的PSAC拮抗剂具有更高特异性和亲和力的化合物。最后，我们探测了添加和不添加拮抗剂的通道门控，以确定PSAC只有一个开放状态和至少三个关闭状态。我们的研究为这个不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.