Roles of Cytoskeletal Components on the Membrane Movement of Individual Bk Ca Channels in Live COS-7 cell

Abstract

Membrane dynamics of the large-conductance Ca2+-activated K+ channels (BKCa channels) was investigated in COS-7 cells by co-expressing the α subunit of the channel tagged with an acceptor peptide for biotin at its extracellular N-terminus and a genetically modified E. coli biotin ligase. We were able to visualize individual BKCa channels that had been biotinylated metabolically and then expressed on to the surface of the cells using quantum dots (QDs) coated with streptavidin. The time-lapse imaging on hundreds of homomeric BKCa channel revealed that the channels in live COS-7 cells exhibited a ‘confined diffusion’ in an area of 1.915 μm2 with an initial diffusion coefficient of 0.033 μm2/s. In order to understand the roles of cytoskeleton on the diffusional characteristics of the channel, we monitored the channel movement in the presence of drugs known to disrupt the filamentous actin and the microtubule, and quantified their effects on channel dynamics. It was found that both diffusion coefficient and the area of diffusion are differentially affected by such drug treatments. We are currently investigating the domain(s) of the channel responsible for cytoskeletal influence on the membrane dynamics of BKCa channel. Using this QD-based, single-molecule tracking technique, we wish to understand the cellular players influencing the diffusional movement of BKCa channel and their mechanism of action. ACKNOWLEDGEMENTS The authors are grateful to the members of the Laboratory of Molecular Neurobiology at GIST for their valuable comments and the Bio-Imaging Research Center at GIST for providing various imaging equipments. This research was supported by the grants from the Bio-Imaging Research Center to C-S Park.