High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient association of pore-forming and auxiliary subunits, lateral diffusion, and clustering with other proteins. However, the relationship between lateral diffusion and function is poorly understood. To approach this problem, we describe how lateral mobility and activity of individual channels in supported lipid membranes can be monitored and correlated using total internal reflection fluorescence (TIRF) microscopy.
This video describes how to use TIRF microscopy to track single ion channels and determine their activity in supported lipid membranes, thereby defining the interplay between lateral membrane movement and channel function. It describes how to prepare the membranes, record the data, and analyze the results.
In contrast to classical electrophysiological methods, no membrane voltages are required to study ion flux through individual channels. Furthermore, the method does not require labeling with fluorescent dyes or molecules that could interfere with the lateral movement of the channels in the membrane.
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For reference, see: Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using TIRF Microscopy. Wang, S. & Nussberger, S. J. Vis. Exp. 192: 1-21, e64970, doi 10.3791/64970 (2023)