The Plasmodial Surface Anion Channel And Malaria Parasite Nutrient Acquisition

2014年，Apicomplexan分子生理学科继续研究疟疾寄生虫感染后红细胞通透性增加的分子基础和生理作用。 在一项研究中，我们研究了一个不寻常的寄生虫多基因家族如何有助于宿主红细胞膜上营养通道的形成。 先前的研究暗示疟原虫表面阴离子通道（PSAC）和clag多基因家族在红细胞表面营养物质和单价离子的摄取增加。 目前该领域的一个重要问题是CLAG蛋白如何促进通道活性。 在一个模型中，这些蛋白质作为激活宿主细胞表面上已经存在的静止通道的酶发挥作用;在另一个模型中，CLAG蛋白直接促进通道的形成，无论是孤立的还是通过与不相关的蛋白质相互作用。 这种不确定性特别重要，因为clag基因与其他生物体中已知的离子通道没有可检测的同源性。 在本报告年度，我们使用蛋白酶检查通道的组成。 虽然具有不同特异性的蛋白酶都在CLAG 3的胞外结构域内裂解，但它们产生不同程度的转运抑制。 胰凝乳蛋白酶诱导的抑制依赖于寄生虫的基因型，与通道诱导的HB 3寄生虫影响到更大的程度比Dd 2克隆。在HB 3xDd 2遗传杂交、DNA转染和基因沉默实验中功能性蛋白水解的遗传都指向clag 3基因，为这些基因的作用提供了独立的证据。Dd 2特异性抑制剂和定点诱变的蛋白酶保护试验表明，CLAG 3细胞外环上的变体L1115 F残基有助于抑制剂结合，并解释了功能性蛋白水解的差异。这些发现表明，表面暴露的CLAG 3直接有助于通道功能;它们还提供了对PSAC孔的早期结构见解。 PLoS ONE 9：e93759（2014）。 在第二项研究中，我们检查了PSAC识别和区分溶质吸收的不寻常能力。 这个问题很重要，因为有许多营养素和抗疟药物主要通过PSAC进入受感染的红细胞。 尽管渗透溶质的范围很广，但通道严格排除钠离子;这种排除对于宿主血浆中细胞内寄生虫的存活至关重要。 在这里，我们探索了这种显着的溶质选择性的机制，并确定胍作为一种有机阳离子，具有高渗透性的红细胞感染疟疾寄生虫，但未感染的细胞可以忽略不计的摄取。 转运特性和药理学表明，这种摄取是由PSAC特异性介导的。 我们还研究了有机和无机阳离子渗透性，并提出阳离子脱水是通过通道运输的限速步骤。受感染细胞的高胍渗透性也允许寄生虫培养物的快速和严格的同步化，如该病原体的分子和细胞研究所需。这项研究提供了一个框架，养分和离子渗透通过PSAC。 了解渗透的结构和分子基础对于了解通道在宿主-寄生虫相互作用中的作用以及开发可能成为未来抗疟疾药物的抑制剂至关重要。 BioMed Research International，in press（2014）. 在第三项研究中，我们检测了感染细胞对钙离子（一种必需的二价阳离子）的渗透性增加。 我们使用非破坏性加载的荧光钙指示剂染料（Fluo-8）到人红细胞定量Ca++摄取动力学。 我们的研究表明，疟疾寄生虫感染引起红细胞Ca++渗透性显着增加。 药理学研究表明，这种摄取不是由PSAC或典型的哺乳动物Ca++通道介导的。 寄生虫生长抑制研究揭示了细胞外Ca++的保守需求。 这些发现表明感染后Ca++摄取的新途径。 该途径的抑制剂可能是抗疟药物开发的极好起点。 Malaria J. 13：184（2014）。

In 2014, the Apicomplexan Molecular Physiology Section continued studies into the molecular basis and physiological role of increased erythrocyte permeability after infection with malaria parasites. In one study, we examined how an unusual parasite multigene family contributes to formation of nutrient channels at the host erythrocyte membrane. Previous studies implicated the plasmodial surface anion channel (PSAC) and the clag multigene family in the increased uptake of nutrients and monovalent ions at the erythrocyte surface. An important question in the field at present is how CLAG proteins contribute to channel activity. In one model, these proteins function as enzymes that activate quiescent channels already present on the host cell surface; in another, the CLAG proteins contribute directly to formation of the channel, either in isolation or through interactions with unrelated proteins. This uncertainly is especially important because the clag genes do not have detectable homology to known ion channels in other organisms. In this reporting year, we used proteases to examine the channel's composition. While proteases with distinct specificities all cleaved within an extracellular domain of CLAG3, they produced differing degrees of transport inhibition. Chymotrypsin-induced inhibition depended on parasite genotype, with channels induced by the HB3 parasite affected to a greater extent than those of the Dd2 clone. Inheritance of functional proteolysis in the HB3xDd2 genetic cross, DNA transfection, and gene silencing experiments all pointed to the clag3 genes, providing independent evidence for a role of these genes. Protease protection assays with a Dd2-specific inhibitor and site-directed mutagenesis revealed that a variant L1115F residue on a CLAG3 extracellular loop contributes to inhibitor binding and accounts for differences in functional proteolysis. These findings suggest that surface-exposed CLAG3 contributes directly to channel function; they also provide early structural insights into the PSAC pore. PLoS ONE 9: e93759 (2014). In a second study, we examined the unusual ability of PSAC to identify and distinguish solutes for uptake. This question is important because there are many nutrients and antimalarial drugs that enter infected erythrocytes primarily via PSAC. Despite the broad range of permeant solutes, the channel stringently excludes sodium ions; this exclusion is essential for survival of the intracellular parasite in host plasma. Here, we explored mechanisms for this remarkable solute selectivity and identified guanidinium as an organic cation with high permeability into erythrocytes infected with malaria parasites, but negligible uptake by uninfected cells. Transport characteristics and pharmacology indicated that this uptake is specifically mediated by PSAC. We also examined organic and inorganic cation permeabilities and proposed that cation dehydration is the rate-limiting step in transport through the channel. The high guanidinium permeability of infected cells also allows rapid and stringent synchronization of parasite cultures, as required for molecular and cellular studies of this pathogen. This study provides a framework for nutrient and ion permeation through PSAC. Understanding the structural and molecular basis of permeation is critical to knowing the channel's role in host-parasite interactions and to developing inhibitors that may be future antimalarial drugs. BioMed Research International, in press (2014). In a third study, we examined the increased permeability of infected cells to calcium, an essential divalent cation. We used nondestructive loading of a fluorescent calcium indicator dye (Fluo-8) into human erythrocytes to quantify Ca++ uptake kinetics. Our studies revealed that infection with malaria parasites produces marked increases in erythrocyte Ca++ permeability. Pharmacological studies revealed that this uptake is not mediated by PSAC or by typical mammalian Ca++ channels. Parasite growth inhibition studies revealed a conserved requirement for extracellular Ca++. These findings suggest a novel pathway for Ca++ uptake after infection. Inhibitors of this pathway may be excellent starting points for antimalarial drug development. Malaria J. 13:184 (2014).