Goh Melvin Wei Shern, Tozawa Yuzuru, Tero Ryugo
Department of Applied Chemistry and Life Science, Toyohashi University of Technology, Toyohashi 441-8580, Japan.
Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.
Membranes (Basel). 2023 Oct 25;13(11):854. doi: 10.3390/membranes13110854.
Artificial lipid bilayer systems, such as vesicles, black membranes, and supported lipid bilayers (SLBs), are valuable platforms for studying ion channels at the molecular level. The reconstitution of the ion channels in an active form is a crucial process in studies using artificial lipid bilayer systems. In this study, we investigated the assembly of the human related gene (hERG) channel prepared in a cell-free synthesis system. AFM topographies revealed the presence of protrusions with a uniform size in the entire SLB that was prepared with the proteoliposomes (PLs) incorporating the cell-free-synthesized hERG channel. We attributed the protrusions to hERG channel monomers, taking into consideration the AFM tip size, and identified assembled structures of the monomer that exhibited dimeric, trimeric, and tetrameric-like arrangements. We observed molecular images of the functional hERG channel reconstituted in a lipid bilayer membrane using AFM and quantitatively evaluated the association state of the cell-free synthesized hERG channel.
人工脂质双层系统，如囊泡、黑膜和支撑脂质双层（SLB），是在分子水平上研究离子通道的重要平台。在使用人工脂质双层系统的研究中，将离子通道重构为活性形式是一个关键过程。在本研究中，我们研究了在无细胞合成系统中制备的人类相关基因（hERG）通道的组装情况。原子力显微镜（AFM）形貌显示，在用掺入无细胞合成hERG通道的蛋白脂质体（PL）制备的整个SLB中存在大小均匀的突起。考虑到AFM针尖尺寸，我们将这些突起归因于hERG通道单体，并确定了呈现二聚体、三聚体和四聚体样排列的单体组装结构。我们使用AFM观察了重构在脂质双层膜中的功能性hERG通道的分子图像，并定量评估了无细胞合成hERG通道的缔合状态。
