Cellular and Molecular Physiology of Bloodstream Malaria Parasites

2023年，Apicomexan分子生理学部分疟疾研究了细胞内人类疟疾寄生虫恶性疟原虫内的Na+和H+动态平衡。这种寄生虫通过PfATP4的作用来维持这种动态平衡，PfATP4是一种定位于细胞内寄生虫质膜的阳离子泵。PfATP4是高级抗疟疾先导的靶标，它会在感染的红细胞内产生许多鲜为人知的代谢紊乱。为了更好地了解这些干扰，我们现在在寄生虫的质膜上表达了哺乳动物配体门控的TRPV1离子通道，以研究离子调节和阳离子泄漏的影响。TRPV1的表达是耐受性良好的，与通过非激活通道的可忽略的离子通量一致。在其激活浓度下，TRPV1配体能使转染系中的寄生虫迅速死亡，但对野生型亲本无害。激活触发了寄生虫质膜上胆固醇的重新分布，复制了PfATP4抑制剂的作用，并直接涉及到这一过程中的阳离子调节失调。与预测相反，在低Na+介质中激活TRPV1可增强对寄生虫的杀灭作用，但PfATP4抑制剂的效果不变。对一个配体抗性突变体的选择揭示了TRPV1中一个以前没有特征的G683V突变，它阻塞了下通道门，这意味着通透性降低是寄生虫对以离子稳态为靶点的抗疟疾药物的抗性机制之一。我们的发现提供了对疟疾寄生虫离子调控的关键见解，并将指导作用于宿主-病原体界面的高级抗疟疾先导的作用机制研究。PLOS One 18：E0283776(2023)。PMID：37014920 在另一项研究中，我们检测了红细胞膜的钙摄取和外流。Ca++是许多细胞发育活动所必需的，也是细胞内疟疾寄生虫所必需的。尽管需要，但通过高效的PMCA Ca++挤压泵，人体红细胞中的Ca++浓度保持在非常低的水平。虽然我们对这种钙离子挤出泵的大部分知识来自于对人红细胞的研究，但对这些细胞钙离子转运的动力学研究仅限于放射性同位素通量测量。在这里，我们开发了一种健壮的、基于微孔板的红细胞钙外流分析方法，使用细胞外荧光钙离子指示剂。我们优化了A23187离子载体的钙负载，建立了去除离子载体的条件，并通过添加胞外EGTA来调整荧光染料的灵敏度，以允许连续跟踪钙离子外流。外流动力学被葡萄糖加速，并被非特异性抑制剂钒酸盐以剂量依赖的方式抑制，这表明钙泵活性可以在384孔微孔板中被跟踪。这些研究使无放射性同位素的钙泵动力学测量成为可能，并应有助于筛选这一基本转运活动的特定抑制剂。欧元。生物群落。J.52：101-110(2023)。PMID：36512028

In 2023, the Apicomplexan Molecular Physiology Section Malaria examined Na+ and H+ homeostasis within the intracellular human malaria parasite, Plasmodium falciparum. This parasite maintains this homeostasis through the action of PfATP4, a cation pump localized to the intracellular parasite plasma membrane. PfATP4 is the target of advanced antimalarial leads, which produce many poorly understood metabolic disturbances within infected erythrocytes. To better understand these disturbances, we have now expressed the mammalian ligand-gated TRPV1 ion channel at the parasite plasma membrane to study ion regulation and examine the effects of cation leak. TRPV1 expression was well-tolerated, consistent with negligible ion flux through the nonactivated channel. TRPV1 ligands produced rapid parasite death in the transfectant line at their activating concentrations, but were harmless to the wild-type parent. Activation triggered cholesterol redistribution at the parasite plasma membrane, reproducing effects of PfATP4 inhibitors and directly implicating cation dysregulation in this process. In contrast to predictions, TRPV1 activation in low Na+ media accentuated parasite killing but a PfATP4 inhibitor had unchanged efficacy. Selection of a ligand-resistant mutant revealed a previously uncharacterized G683V mutation in TRPV1 that occludes the lower channel gate, implicating reduced permeability as a mechanism for parasite resistance to antimalarials targeting ion homeostasis. Our findings provide key insights into malaria parasite ion regulation and will guide mechanism-of-action studies for advanced antimalarial leads that act at the host-pathogen interface. PLoS One 18:e0283776 (2023). PMID: 37014920 In another study, we examined Ca++ uptake and efflux at the erythrocyte membrane. Ca++ is required for numerous cellular developmental activities and is required by intracellular malaria parasites. Despite its requirement, Ca++ is maintained at very low concentrations in human erythrocytes by an efficient PMCA Ca++ extrusion pump. Although much of our knowledge about this Ca++ extrusion pump derives from studies with human erythrocytes, kinetic studies of Ca++ transport for these cells are limited to radioisotope flux measurements. Here, we developed a robust, microplate-based assay for erythrocyte Ca++ efflux using extracellular fluorescent Ca++ indicators. We optimized Ca++ loading with the A23187 ionophore, established conditions for removal of the ionophore, and adjusted fluorescent dye sensitivity by addition of extracellular EGTA to allow continuous tracking of Ca++ efflux. Efflux kinetics were accelerated by glucose and inhibited in a dose-dependent manner by the nonspecific inhibitor vanadate, revealing that Ca++ pump activity can be tracked in a 384-well microplate format. These studies enable radioisotope-free kinetic measurements of the Ca++ pump and should facilitate screens for specific inhibitors of this essential transport activity. Eur. Biophys. J. 52:101-110 (2023). PMID: 36512028