Dynamics of single potassium channel proteins in the plasma membrane of migrating cells

Abstract

Cell migration is an important physiological process among others controlled by ion channel activity. Calcium-activated potassium channels (KCa3.1) are required for optimal cell migration. Previously, we identified single human (h)KCa3.1 channel proteins in the plasma membrane by means of quantum dot (QD) labeling. In the present study, we tracked single-channel proteins during migration to classify their dynamics in the plasma membrane of MDCK-F cells. Single hKCa3.1 channels were visualized with QD- or Alexa488-conjugated antibodies and tracked at the basal cell membrane using time-lapse total internal reflection fluorescence (TIRF) microscopy. Analysis of the trajectories allowed the classification of channel dynamics. Channel tracks were compared with those of free QD-conjugated antibodies. The size of the label has a pronounced effect on hKCa3.1 channel diffusion. QD-labeled channels have a (sub)diffusion coefficient DQDbound= 0.067 μm2/sα, whereas that of Alexa488-labeled channels is DAlexa= 0.139 μm2/s. Free QD-conjugated antibodies move much faster: DQDfree= 2.163 μm2/sα. Plotting the mean squared distances (msd) covered by hKCa3.1 channels as a function of time points to the mode of diffusion. Alexa488-labeled channels diffuse normally, whereas the QD-label renders hKCa3.1 channel diffusion anomalous. Free QD-labeled antibodies also diffuse anomalously. Hence, QDs slow down diffusion of hKCa3.1 channels and change the mode of diffusion. These results, referring to the role of label size and properties of the extracellular environment, suggest that the pericellular glycocalyx has an important impact on labels used for single molecule tracking. Thus tracking fluorescent particles within the glycocalyx opens up a possibility to characterize the pericellular nanoenvironment.