Dynamics of Individual BKCa Channels in Live Cells Monitored by Site-Specific Labeling Using Quantum Dots

Abstract

In order to monitor the movement of individual large-conductance Ca2+-activated K+ channels (BKCa channels) in live cells at real-time, we co-expressed the BKCa channel tagged at its extracellular N-terminus with the ‘acceptor peptide (AP)’ for biotin and a genetically modified E. coli biotin ligase in various mammalian cells. Using the quantum dots (QDs) coated with streptavidin, we were able to visualize individual BKCa channels that had been biotinylated intracellularly and then expressed on to the surface of the cells. The channels were determined to be labeled by two QDs in average, based on the levels of quantized fluctuations of fluorescence intensity, known as ‘photo-blinkings’. We monitored the movements of BKCa channels in both live mammalian cell-lines and primary hippocampal neurons using time-lapse imaging. Depending on the type of cells and the location where the channels were expressed within a cell, BKCa channels showed different patterns and speeds in their movements. We are currently quantifying the movement of individual channels and investigating those protein motifs affecting the channel dynamics. We wish to understand the molecular mechanism of BKCa channel trafficking and the cellular players involved in.