Assembly of Cell-Free Synthesized Ion Channel Molecules in Artificial Lipid Bilayer Observed by Atomic Force Microscopy

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 ether-a-go-go-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.