Oriented Reconstitution of a Membrane Protein in a Giant Unilamellar Vesicle: Experimental Verification with the Potassium Channel KcsA,Journal of the American Chemical Society

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Oriented Reconstitution of a Membrane Protein in a Giant Unilamellar Vesicle: Experimental Verification with the Potassium Channel KcsA
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2011-08-03 , DOI: 10.1021/ja2040859
Miho Yanagisawa ₁ , Masayuki Iwamoto ₂ , Ayako Kato _{1,

3} , Kenichi Yoshikawa ₁ , Shigetoshi Oiki ₂

Affiliation

We report a method for the successful reconstitution of the KcsA potassium channel with either an outside-out or inside-out orientation in giant unilamellar vesicles, using the droplet-transfer technique. The procedure is rather simple. First, we prepared water-in-oil droplets lined with a lipid monolayer. When solubilized KcsA was encapsulated in the droplet, it accumulated at monolayers of phosphatidylglycerol (PG) and phosphoethanolamine (PE) but not at a monolayer of phosphatidylcholine (PC). The droplet was then transferred through an oil/water interface having a preformed monolayer. The interface monolayer covered the droplet so as to generate a bilayer vesicle. By creating chemically different lipid monolayers at the droplet and oil/water interface, we obtained vesicles with asymmetric lipid compositions in the outer and inner leaflets. KcsA was spontaneously inserted into vesicles from the inside or outside, and this was accelerated in vesicles that contained PE or PG. Integrated insertion into the vesicle membrane and the KcsA orientation were examined by functional assay, exploiting the pH sensitivity of the opening of the KcsA when the pH-sensitive cytoplasmic domain (CPD) faces toward acidic media. KcsA loaded from the inside of the PG-containing vesicles becomes permeable only when the intravesicular pH is acidic, and the KcsA loaded from the outside becomes permeable when the extravesicular pH is acidic. Therefore, the internal or external insertion of KcsA leads to an outside-out or inside-out configuration so as to retain its hydrophilic CPD in the added aqueous side. The CPD-truncated KcsA exhibited a random orientation, supporting the idea that the CPD determines the orientation. Further application of the droplet-transfer method is promising for the reconstitution of other types of membrane proteins with a desired orientation into cell-sized vesicles with a targeted lipid composition of the outer and inner leaflets.

中文翻译：

巨大单层囊泡中膜蛋白的定向重建：钾通道 KcsA 的实验验证

我们报告了一种使用液滴转移技术成功重建 KcsA 钾通道的方法，该通道在巨型单层囊泡中具有由外向外或由内向外的方向。程序比较简单。首先，我们制备了内衬脂质单层的油包水液滴。当溶解的 KcsA 被包裹在液滴中时，它会在单层磷脂酰甘油 (PG) 和磷酸乙醇胺 (PE) 处积累，但不会在单层磷脂酰胆碱 (PC) 处积累。然后将液滴转移通过具有预先形成的单层的油/水界面。界面单层覆盖液滴以产生双层囊泡。通过在液滴和油/水界面处产生化学上不同的脂质单层，我们获得了在外部和内部小叶中具有不对称脂质成分的囊泡。KcsA 从内部或外部自发地插入囊泡中，这在含有 PE 或 PG 的囊泡中加速。当 pH 敏感细胞质结构域 (CPD) 面向酸性介质时，利用 KcsA 开口的 pH 敏感性，通过功能测定检查整合插入囊泡膜和 KcsA 方向。只有当囊泡内的 pH 值呈酸性时，从含 PG 囊泡内部加载的 KcsA 才具有渗透性，而当囊泡外的 pH 值呈酸性时，从外部加载的 KcsA 才具有渗透性。因此，KcsA 的内部或外部插入导致外向或由内向外的构型，以便在添加的水侧保持其亲水性 CPD。CPD 截断的 KcsA 表现出随机取向，支持 CPD 决定方向的想法。液滴转移方法的进一步应用有望将具有所需方向的其他类型的膜蛋白重组为细胞大小的囊泡，并具有外小叶和内小叶的靶向脂质成分。

更新日期：2011-08-03

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