The Plasmodial Surface Anion Channel And Malaria Parasite Nutrient Acquisition

2015年，Apicomplexan分子生理学部分研究了感染疟疾寄生虫P. falciparum的人类红细胞吸收营养和毒素的分子基础。 我们先前使用体外选择产生HB 3-leuR 1，一种对亮抑酶肽毒素具有抗性的寄生虫系。 这种蛋白酶抑制剂被认为是膜不渗透的，但必须进入感染的细胞才能杀死寄生虫。 HB 3-LeuR 1不仅表现出对亮抑酶肽的摄取减少，而且表现出对通过疟原虫表面阴离子通道（PSAC）进入感染细胞的其他溶质的摄取减少，PSAC是我们小组鉴定的一种宽渗透性营养和离子通道。 在clag 3基因中有一个与PSAC活性相关的突变，但这种非同义突变（A1210 T）、PSAC活性改变和亮抑酶素耐药之间的关系尚不清楚。 在目前的研究中，我们使用计算研究，以确定一个保守的两亲性结构域，包括A1210残基的CLAG 3蛋白。 螺旋轮分析揭示了极性和疏水残基的严格隔离的相对面的推定的α-螺旋，一致的跨膜结构域线的水填充孔。 因此，我们使用野生型疟疾寄生虫的定点诱变和DNA转染来引入A1210 T突变而不进行亮抑酶肽选择。 转染的寄生虫表现出改变溶质的选择性，通过PSAC，通道药理学的变化，并增加耐受亮抑酶素的挑战。 这些发现支持CLAG 3对PSAC活性和有机溶质转运的直接贡献。 它们还揭示了一种新的抗疟耐药机制的分子基础，该机制涉及减少通道介导的毒素和药物摄取。 这些发现提供了对PSAC结构-功能的深入了解，并应告知抗疟药物开发计划获得性耐药的风险。 感染和免疫83（6）：2566-74。（2015年）。

In 2015, the Apicomplexan Molecular Physiology Section examined the molecular basis of nutrient and toxin uptake by human erythrocytes infected with the malaria parasite, P. falciparum. We previously used in vitro selections to generate HB3-leuR1, a parasite line resistant to the toxin leupeptin. This protease inhibitor is considered membrane-impermeant, but must enter infected cells to kill the parasite. HB3-LeuR1 exhibited reduced uptake of not only leupeptin but other solutes that enter infected cells via the plasmodial surface anion channel (PSAC), a broad permeability nutrient and ion channel identified by our group. There was a single mutation in the clag3 gene linked to PSAC activity, but the relationship between this nonsynonymous mutation (A1210T), altered PSAC activity, and leupeptin resistance was unclear. In the present studies, we used computational studies to identify a conserved amphipathic domain including the A1210 residue on the CLAG3 protein. Helical wheel analysis revealed strict segregation of polar and hydrophobic residues to opposite faces of the putative alpha-helix, consistent with a transmembrane domain that lines a water-filled pore. We therefore used site-directed mutagenesis and DNA transfection of wild-type malaria parasites to introduce the A1210T mutation without leupeptin selection. The transfected parasite exhibited altered solute selectivity through PSAC, changes in channel pharmacology, and increased tolerance to leupeptin challenge. These findings support a direct contribution of CLAG3 to PSAC activity and organic solute transport. They also reveal the molecular basis of a novel antimalarial resistance mechanism that involves reduced channel-mediated uptake of toxins and drugs. These findings provide insights into PSAC structure-function and should inform antimalarial drug development programs about the risk of acquired resistance. Infection and Immunity 83(6):2566-74. (2015).