Zheng Hui, Lee Sungsoo, Llaguno Marc C, Jiang Qiu-Xing
Department of Cell Biology, Department of Physiology, and Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, TX 75390 Department of Cell Biology, Department of Physiology, and Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, TX 75390.
Department of Cell Biology, Department of Physiology, and Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, TX 75390 Department of Cell Biology, Yale University, New Haven, CT 06510.
J Gen Physiol. 2016 Jan;147(1):77-93. doi: 10.1085/jgp.201511448.
Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid composition of membranes. Here, using a newly developed chemical engineering procedure, we report on a bead-supported unilamellar membrane (bSUM) system that allows good control over membrane dimension, protein orientation, and lipid composition. Our new system uses specific ligands to facilitate the unidirectional incorporation of membrane proteins into lipid bilayers. Cryo-electron microscopic imaging demonstrates the unilamellar nature of the bSUMs. Electrical recordings from voltage-gated ion channels in bSUMs of varying diameters demonstrate the versatility of the new system. Using KvAP as a model system, we show that compared with other in vitro membrane systems, the bSUMs have the following advantages: (a) a major fraction of channels are orientated in a controlled way; (b) the channels mediate the formation of the lipid bilayer; (c) there is one and only one bilayer membrane on each bead; (d) the lipid composition can be controlled and the bSUM size is also under experimental control over a range of 0.2-20 µm; (e) the channel activity can be recorded by patch clamp using a planar electrode; and (f) the voltage-clamp speed (0.2-0.5 ms) of the bSUM on a planar electrode is fast, making it suitable to study ion channels with fast gating kinetics. Our observations suggest that the chemically engineered bSUMs afford a novel platform for studying lipid-protein interactions in membranes of varying lipid composition and may be useful for other applications, such as targeted delivery and single-molecule imaging.
融合或巨型囊泡、平面脂质双层、液滴膜系统和平面膜支撑体系已被开发用于整合膜蛋白,以便对这类蛋白进行电学和生物物理分析或研究双层膜的性质。然而,将包括离子通道在内的膜蛋白整合到重构膜系统中仍然存在困难,这些系统需要能够轻松控制操作尺寸、膜蛋白的整合方向以及膜的脂质组成。在此,我们使用一种新开发的化学工程方法,报道了一种珠子支撑的单层膜(bSUM)系统,该系统能够很好地控制膜尺寸、蛋白方向和脂质组成。我们的新系统使用特定配体促进膜蛋白单向整合到脂质双层中。冷冻电子显微镜成像证明了bSUM的单层性质。对不同直径的bSUM中电压门控离子通道的电记录证明了新系统的多功能性。以KvAP作为模型系统,我们表明与其他体外膜系统相比,bSUM具有以下优点:(a)大部分通道以可控方式定向;(b)通道介导脂质双层的形成;(c)每个珠子上只有一层双层膜;(d)脂质组成可以控制,bSUM大小在0.2 - 20 µm范围内也可通过实验控制;(e)通道活性可以使用平面电极通过膜片钳记录;(f)平面电极上bSUM的电压钳制速度(0.2 - 0.5毫秒)很快,使其适合研究具有快速门控动力学的离子通道。我们的观察结果表明,化学工程改造的bSUM为研究不同脂质组成膜中的脂 - 蛋白相互作用提供了一个新平台,并且可能对其他应用有用,如靶向递送和单分子成像。