Functional reconstitution into liposomes of purified human RhCG ammonia channel.
Functional reconstitution into liposomes of purified human RhCG ammonia channel.
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DOI：
10.1371/journal.pone.0008921
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发表时间：
2010-01-28
期刊：
影响因子：
3.7
通讯作者：
Ripoche P
中科院分区：
文献类型：
--
作者：
Mouro-Chanteloup I;Cochet S;Chami M;Genetet S;Zidi-Yahiaoui N;Engel A;Colin Y;Bertrand O;Ripoche P
Rh 糖蛋白（RhAG、RhBG、RhCG）是 Amt/Mep/Rh 家族的成员，可促进铵跨质膜的移动。已经在不同的异源系统（例如酵母、卵母细胞和真核细胞系）中描述了 Rh 糖蛋白表达后铵转运活性的变化。然而，在这些复杂的系统中，不能排除内源蛋白对此功能的潜在贡献。为了证明 Rh 糖蛋白本身可以转运 NH3，将人 RhCG 纯化至同质并重构为脂质体，从而对其通道功能特性提供了新的见解。使用在 RhCG 的第二个胞外环中引入的 HA 标签来纯化来自去污剂溶解的重组 HEK293E 细胞的 HA 标签的 RhCG 糖蛋白，使其均质。负染纯化 RhCG-HA 的电子显微镜分析显示，经过图像处理后，形成具有三聚蛋白结构的直径 9 nm 的均匀颗粒。在 C12E8 去垢剂存在下用鞘磷脂、磷脂酰胆碱和磷脂酸脂质进行重构，随后通过 Biobeads 将其去除。通过冷冻断裂电子显微镜控制蛋白质掺入。脂质体中的颗粒密度是脂质/蛋白质比率的函数。与空脂质体相比，RhCG-蛋白脂质体中的铵渗透性增加了两倍和三倍，具体取决于脂质/蛋白质比率（分别为 1/300 和 1/150）。这种强烈的 NH3 运输被汞盐和铜盐可逆地抑制，并表现出较低的阿伦尼乌斯活化能。这项研究允许测定每个 RhCG 单体的氨渗透性，表明表观 PunitNH3（约 1×10−3 µm3.s−1）接近表达重组人 RhCG 的 HEK293E 细胞（1.60×10−3 µm3.s−1）和内源表达 RhAG 的人红细胞（2.18×10−3）中测量的渗透性 µm3.s−1)。这项研究的主要发现是 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。
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 NH3, human RhCG was purified to homogeneity and reconstituted into liposomes, giving new insights into its channel functional properties. 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 C12E8 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 NH3 transport was reversibly inhibited by mercuric and copper salts and exhibited a low Arrhenius activation energy. This study allowed the determination of ammonia permeability per RhCG monomer, showing that the apparent PunitNH3 (around 1×10−3 µm3.s−1) is close to the permeability measured in HEK293E cells expressing a recombinant human RhCG (1.60×10−3 µm3.s−1), and in human red blood cells endogenously expressing RhAG (2.18×10−3 µm3.s−1). The major finding of this study is that RhCG protein is active as an NH3 channel and that this function does not require any protein partner.
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影响因子：
5.6
作者：
Lee, Seok-Yong;Letts, James A.;MacKinnon, Roderick
通讯作者：
MacKinnon, Roderick
DOI：
10.1073/pnas.0507886102
发表时间：
2005-10-25
影响因子：
11.1
作者：
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作者：
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DOI：
10.1152/ajprenal.00147.2005
发表时间：
2006-02-01
影响因子：
4.2
作者：
Mak, DOD;Dang, B;Westhoff, CM
通讯作者：
Westhoff, CM