a b s t r a c t BK Ca channels are palmitoylated at a cluster of cysteine residues within the cytosolic linker connecting the 1st and 2nd transmembrane domains, and this lipid modification affects their surface expression. To verify the effects of palmitoylation on the diffusional dynamics of BK Ca channels, we investigated their lateral movement. Compared to wild-type channels, the movement of mutant palmitoylation-deficient channels was much less confined and close to random. The diffusion of the mutant channel was also much faster than that of the wild type. Thus, the lateral movement of BK Ca channels is greatly influenced by palmitoylation.
V-set and transmembrane domain-containing protein 5 (Vstm5), a newly characterized small membrane glycoprotein, can induce membrane protrusions in various cells. Vstm5 can modulate both the position and complexity of central neurons by altering their membrane morphology and dynamics. In this study, we investigated the significance of glycosylation in the expression and function of Vstm5. Four N-linked glycosylation sites (Asn43, Asn87, Asn101, and Asn108) are predicted to be located in the extracellular N-terminus of mouse Vstm5. Although all four sites were glycosylated, their functional roles may not be identical. N-glycosylation at multiple sites affects differentially the function of Vstm5. Glycosylation at individual sites not only played essential roles in surface expression of Vstm5 but also in the formation of neuronal dendritic filopodia. These results indicate that N-linked glycosylation at multiple sites plays important roles by differentially influencing the expression, targeting, and biological activity of Vstm5.
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.
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