Time-lapse Imaging of Individual BKCa Channels in Live Cells Using Site-specific Labeling of Quantum Dots

Abstract

Although the proper localization of specific ion channels at certain regions of cell membrane is essential for their cellular functions, it is a great challenge to visualize and to trace individual channel proteins in live cells. We utilized quantum dots (QDs) to label the large-conductance Ca2+-activated K+ channels (BKCa channels) and monitored their movement in real-time. A site-specific biotinylation was achieved by genetically inserting the ‘acceptor peptide’ sequence at the extracellular N-terminus of the channel and by co-expressing the channel with the E. coli biotin-ligase modified to target into endoplasmic reticulum. After brief incubation of streptavidin-conjugated QDs, strong cell surface labeling of QDs was detected in both COS7 cell and cultured hippocampal pyramidal neurons. By tracking the labeled QDs using time-lapse imaging, we were able to monitor single BKCa channels with high resolution in live cells. In addition, two-color pulse-chase labeling allowed us to observe the channel trafficking to cell surface membrane de novo and their redistribution in real-time. Using the time-lapse imaging of QD-labeled channel protein as an assay system, we were able to show the differential roles of cytoskeletons in trafficking and dynamics of BKCa channels. This new approach can be applied to study the cellular behaviors of ion channels in many different aspects.