Building “Leaning Towers” on Living Cell Membrane with Single Artificial Channel Molecules

Aquaporins (AQPs) are essential channel proteins that regulate water and small ions transport across biological membranes. In this study, we investigate the three-dimensional (3D) conformation and dynamics of artificial aquaporins (AAQPs) on living cell membranes at the single-molecule level using defocused fluorescence imaging. By selectively labeling AAQPs with fluorescent dyes, we directly visualize their orientation and rotational motions on the cell membrane. Our results reveal that AAQPs exhibit a tilted conformation on the negatively charged cell membrane, with polar angles predominantly less than 45°, which is driven by electrostatic interactions between the positively charged terminal of AAQPs and the lipid membrane. The rotational motions of AAQPs are constrained, particularly in the vertical direction, owing to interactions with the extracellular matrix. In contrast, AAQPs with negatively charged terminals show reduced penetration efficiency and faster rotational fluctuations, highlighting the critical role of electrostatic interactions in pore formation. These findings provide fundamental insights into the structural and dynamic behaviors of artificial channel molecules on living cell membranes, offering valuable guidance for the design of functional synthetic channels.