Effects of Palmitoylation on the Movement of BKCA Channels In Live Cells using Site-Specific Labeling with Quantum Dots

Abstract

It was shown that the large-conductance Ca 2þ -activated K þ (BK Ca ) channels are palmioylated at a cluster of cysteine residues within the cytosolic linker connecting the 1 st and 2 nd transmembrane domains,and the lipid modification affects the surface expression of the channel proteins. Since the lipid modification is known to influence the dynamics of the membrane proteins, we investigated the effect of palmitoylation on the lateral movement of BK Ca channels within the live cell membrane. The wild-type channel and a triple mutant (C53:54:56A) in which three cysteine residues were substituted to alanine residues were tagged with an acceptor peptide sequence at their N-terminus and expressed in COS-7 cells for metabolic modification by endogenous biotin. The biotin-modified BK Ca channels presented on the cell surface were specifically labeled with streptavidin-conjugated quantum dots (QDs). The QDlabeled BK Ca channels were visualized in live cells and tracked at real-time. Unlike the wild-type channels exhibiting a confined diffusion, the movement of the mutant channels was much less confined and close to be random. The diffusion of the mutant channel was also much faster than that of the wild type. Thus, the lateral movement of BK Ca channelsin live cell membrane is greatly influenced by lipid modification.