INSERM UMR_S 665, Paris, France.
PLoS One. 2010 Jan 28;5(1):e8921. doi: 10.1371/journal.pone.0008921.
Rh glycoproteins (RhAG, RhBG, RhCG) are members of the Amt/Mep/Rh family which facilitate movement of ammonium across plasma membranes. Changes in ammonium transport activity following expression of Rh glycoproteins have been described in different heterologous systems such as yeasts, oocytes and eukaryotic cell lines. However, in these complex systems, a potential contribution of endogenous proteins to this function cannot be excluded. To demonstrate that Rh glycoproteins by themselves transport NH(3), human RhCG was purified to homogeneity and reconstituted into liposomes, giving new insights into its channel functional properties.
METHODOLOGY/PRINCIPAL FINDINGS: An HA-tag introduced in the second extracellular loop of RhCG was used to purify to homogeneity the HA-tagged RhCG glycoprotein from detergent-solubilized recombinant HEK293E cells. Electron microscopy analysis of negatively stained purified RhCG-HA revealed, after image processing, homogeneous particles of 9 nm diameter with a trimeric protein structure. Reconstitution was performed with sphingomyelin, phosphatidylcholine and phosphatidic acid lipids in the presence of the C(12)E(8) detergent which was subsequently removed by Biobeads. Control of protein incorporation was carried out by freeze-fracture electron microscopy. Particle density in liposomes was a function of the Lipid/Protein ratio. When compared to empty liposomes, ammonium permeability was increased two and three fold in RhCG-proteoliposomes, depending on the Lipid/Protein ratio (1/300 and 1/150, respectively). This strong NH(3) transport was reversibly inhibited by mercuric and copper salts and exhibited a low Arrhenius activation energy.
CONCLUSIONS/SIGNIFICANCE: This study allowed the determination of ammonia permeability per RhCG monomer, showing that the apparent Punit(NH3) (around 1x10(-3) microm(3)xs(-1)) is close to the permeability measured in HEK293E cells expressing a recombinant human RhCG (1.60x10(-3) microm(3)xs(-1)), and in human red blood cells endogenously expressing RhAG (2.18x10(-3) microm(3)xs(-1)). The major finding of this study is that RhCG protein is active as an NH(3) channel and that this function does not require any protein partner.
Rh 糖蛋白（RhAG、RhBG、RhCG）是 Amt/Mep/Rh 家族的成员，可促进铵跨质膜的转运。在不同的异源系统（如酵母、卵母细胞和真核细胞系）中，表达 Rh 糖蛋白后，铵转运活性发生了变化。然而，在这些复杂的系统中，不能排除内源性蛋白对该功能的潜在贡献。为了证明 Rh 糖蛋白本身可以转运 NH3，我们将人 RhCG 纯化至均相，并重新构建到脂质体中，从而深入了解其通道功能特性。
方法/主要发现：在 RhCG 的第二个细胞外环中引入 HA 标签，用于从去污剂溶解的重组 HEK293E 细胞中纯化 HA 标记的 RhCG 糖蛋白至均相。对负染纯化的 RhCG-HA 进行电子显微镜分析，经过图像处理后，得到直径为 9nm 的均相颗粒，具有三聚体蛋白结构。用鞘磷脂、卵磷脂和磷脂酸脂质与 C12E8 去污剂一起进行重建，然后用 Biobeads 去除去污剂。通过冷冻断裂电子显微镜控制蛋白掺入。脂质体中的蛋白密度是脂质/蛋白比的函数。与空脂质体相比，取决于脂质/蛋白比（分别为 1/300 和 1/150），在 RhCG-载脂蛋白脂质体中，铵通透性增加了 2 倍和 3 倍。这种强烈的 NH3 转运可被汞盐和铜盐可逆抑制，并表现出低 Arrhenius 活化能。
结论/意义：本研究确定了 RhCG 单体的氨通透性，表明表观 Punit(NH3)（约 1x10(-3) µm3xs(-1)）接近在表达重组人 RhCG 的 HEK293E 细胞中测量的值（1.60x10(-3) µm3xs(-1)），并且在人红细胞中表达 RhAG（2.18x10(-3) µm3xs(-1)）。本研究的主要发现是 RhCG 蛋白作为 NH3 通道具有活性，并且该功能不需要任何蛋白伴侣。
