The role of lateral diffusion of proteins in the membrane in the context of function has not been examined extensively. The data set addresses the relationship between protein lateral diffusion and channel activity of the general protein import pore of mitochondria (TOM-CC). Optical ion flux sensing through single TOM-CC molecules shows that TOM-CC can occupy three ion permeability states. Whereas freely diffusing TOM-CC molecules are preferentially found in a high permeability state, physical tethering to an agarose support causes the channels to transition to intermediate and low permeability states. This data shows that combinatorial opening and closing of the two pores of TOM-CC correlates with lateral protein diffusion in the membrane plane, and that the complex has mechanosensitive-like properties. This is the first demonstration of beta-barrel protein mechanosensitivity, and has direct conceptual consequences for the understanding of the process of mitochondrial protein import. The approach provides a novel tool to simultaneously study the interplay of membrane protein diffusion and channel dynamics.

Dataset of raw video images in Nikon NIS-Elements ND2 (.nd2) image format; filename list of raw video images with spatial single molecule coordinates in Excel (.xlsx) and tabular data (*.tab) format.