The human innate immunity factor apolipoprotein L-I (APOL1) protects against infection by several protozoan parasites, including Trypanosoma brucei brucei. Endocytosis and acidification of high-density lipoprotein–associated APOL1 in trypanosome endosomes leads to eventual lysis of the parasite due to increased plasma membrane cation permeability, followed by colloid-osmotic swelling. It was previously shown that recombinant APOL1 inserts into planar lipid bilayers at acidic pH to form pH-gated nonselective cation channels that are opened upon pH neutralization. This corresponds to the pH changes encountered during endocytic recycling, suggesting APOL1 forms a cytotoxic cation channel in the parasite plasma membrane. Currently, the mechanism and domains required for channel formation have yet to be elucidated, although a predicted helix-loop-helix (H-L-H) was suggested to form pores by virtue of its similarity to bacterial pore-forming colicins. Here, we compare recombinant human and baboon APOL1 orthologs, along with interspecies chimeras and individual amino acid substitutions, to identify regions required for channel formation and pH gating in planar lipid bilayers. We found that whereas neutralization of glutamates within the H-L-H may be important for pH-dependent channel formation, there was no evidence of H-L-H involvement in either pH gating or ion selectivity. In contrast, we found two residues in the C-terminal domain, tyrosine 351 and glutamate 355, that influence pH gating properties, as well as a single residue, aspartate 348, that determines both cation selectivity and pH gating. These data point to the predicted transmembrane region closest to the APOL1 C terminus as the pore-lining segment of this novel channel-forming protein.

中文翻译：

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先天免疫因子APOL1的阳离子通道电导和pH门控由C末端域内的孔衬残基控制。

人类先天免疫因子载脂蛋白LI（APOL1）可防止几种原生动物寄生虫感染，包括布鲁氏锥虫（Trypanosoma brucei brucei）。锥虫内体中高密度脂蛋白相关的APOL1的胞吞作用和酸化会导致寄生虫的最终溶解，这是由于质膜阳离子渗透性增加，随后是胶体渗透溶胀。先前显示，重组APOL1在酸性pH下插入平面脂质双层中，形成pH门控的非选择性阳离子通道，该通道在pH中和后打开。这对应于内吞再循环过程中遇到的pH值变化，表明APOL1在寄生虫质膜中形成了细胞毒性阳离子通道。目前，尚未阐明通道形成所需的机制和域，尽管建议的螺旋-环-螺旋（HLH）由于其与细菌成孔的大肠菌素相似而形成孔。在这里，我们比较了重组人和狒狒的APOL1直系同源物，以及种间嵌合体和单个氨基酸取代，以鉴定通道形成和平面脂质双层中pH门控所需的区域。我们发现，虽然HLH中的谷氨酸盐的中和对于pH依赖性通道的形成可能很重要，但没有证据表明HLH参与pH门控或离子选择性。相比之下，我们在C末端结构域中发现了两个残基，酪氨酸351和谷氨酸355，它们影响pH选通特性，还有一个残基，即天冬氨酸348，它决定了阳离子选择性和pH选通。