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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

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

¹Institute of Physiology II, University of Münster, Münster; and ²Institute of Physiology, Medical Faculty Carl Gustav Carus, Technical University of Dresden, Dresden, Germany

Submitted 13 June 2007 ; accepted in final form 19 February 2008

Cell migration is an important physiological process among others controlled by ion channel activity. Calcium-activated potassium channels (K_Ca3.1) are required for optimal cell migration. Previously, we identified single human (h)K_Ca3.1 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 hK_Ca3.1 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 hK_Ca3.1 channel diffusion. QD-labeled channels have a (sub)diffusion coefficient D_QDbound = 0.067 µm²/s, whereas that of Alexa488-labeled channels is D_Alexa = 0.139 µm²/s. Free QD-conjugated antibodies move much faster: D_QDfree = 2.163 µm²/s. Plotting the mean squared distances (msd) covered by hK_Ca3.1 channels as a function of time points to the mode of diffusion. Alexa488-labeled channels diffuse normally, whereas the QD-label renders hK_Ca3.1 channel diffusion anomalous. Free QD-labeled antibodies also diffuse anomalously. Hence, QDs slow down diffusion of hK_Ca3.1 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.

calcium-activated potassium channel 3.1; quantum dot; single molecule tracking; cell migration