Single molecule imaging of BK channels using total internal reflection fluorescence microscopy

Large-conductance Ca2+-activated K+ (BK) channel has a pivotal role for the regulation of membrane excitability in various types of cells such as smooth muscles, neurons, and secreting organs. The molecular complex and their dynamics of BK channel subunits in the plasma membrane are key issues for understanding of their physiological functions. Here we examined single-molecule imaging of BK channel in the plasma membrane using total internal reflection fluorescence (TIRF) microscopy. Membrane currents were recorded in whole cell clamp configuration. The a-subunit of BK channel was fluorescent-labeled by fusion of the yellow-fluorescent protein (YFP) in either N or C terminus (BK alpha-YFP) and was expressed transiently in human embryo kidney 293 (HEK) cells. The sum of cc-subunit fluorescent images occupied approximately 5 % of the plasma membrane, where their dynamic mobilizations were detected at the level of individual channel unit or their cluster with a diffusion coefficient of approximately 0.01 mu m2/s. When BKa-YFP was co-expressed with BK channel PI-subunit fused with the cyan-fluorescent protein (CFP) (BK beta 1-CFP) in HEK cells, the molecular mobility of the BK channel was reduced to one-fifth. In contrast, the dynamics of the BK alpha-YFP expressed in primary cultured cells of aortic smooth muscle was much more restricted in comparison with that of BK alpha-YFP/beta 1-CFP co-expressed in HEK cells. These results strongly suggest that the mobilization of the cc-subunit is strongly affected by its accessory P-subunit and also the endogenous cytoskeleton. The single-molecule visualization provides us useful information for the in-depth elucidation of molecular complex underlying physiological functions of BK channels.