Abstract
Large-conductance Ca2+-activated K+ (BK) channel has pivotal roles in the regulation of smooth muscle tone. Here we examined single-molecule imaging of BK channel subunits in the plasma membrane using total internal reflection fluorescence (TIRF) microscopy. BK channel α subunit (BKα-YFP) and β1 subunit (BKβ1-CFP) were expressed transiently in cultured rat aortic myocytes or HEK cells. When aortic myocytes were transfected with BKα-YFP, the fluorescent signals of individual channel unit or their clusters were not uniformly distributed in the TIRF images and the sum of fluorescent areas occupied approximately 1% of the plasma membrane in the image. Dynamic mobilizations of BKα-YFP were detected at the levels of individual channel unit and their cluster in HEK cells. When BKα-YFP was coexpressed with BKβ1-CFP in HEK cells, the molecular mobility of the complex was reduced to half of BKα-YFP alone. In contrast, the dynamics of BKα-YFP expressed in aortic myocytes was much more restricted compared with that of BKα-YFP/BKβ1-CFP coexpressed in HEK cells. And actin disruption by 1 μM cytochalasin D in aortic myocytes caused the increase in diffusion coefficient of BKα-YFP. In aortic myocytes, BKα-YFP was co-distributed with caveolin-1 (Cv1-CFP), a marker of caveolae. These results suggest that the mobilization of BK channel α subunit in plasma membrane is strongly restricted or regulated by its accessory β subunit, the cytoskeleton, and the raft structure of membrane surface in native cells such as vascular smooth muscle cells. [J Physiol Sci. 2008;58 Suppl:S76]
