Figure 5.

N-glycosylation regulates the surface expression of hTREK-1 channels. (a) EGFP-reporter coupled WT hTREK-1 channel subunits or glutamine mutants lacking either one or both N-glycosylation motifs were expressed in HeLa cells. Cell membranes stained with Alexa Fluor 594-labeled wheat germ agglutinin are depicted in red. The fluorescence signals of hTREK-1-eGFP variants are shown in green. The overlays (yellow) demonstrate colocalization of diglycosylated, monoglycosylated and nonglycosylated double-mutant channels with the cellular membrane, and show the preserved surface trafficking of deglycosylated channels. Scale bar: 10 µm. (b) Surface fractions of HEK-293T cells expressing the indicted hTREK-1 N-glycosylation-deficient variants were isolated via surface protein biotinylation, followed by streptavidin precipitation. Immunoblots of the input fractions are displayed on the left, and the mean immunosignals of the surface fractions are given on the right side (n = 3). (c) Ion channel subunit surface fractions (i.e., mean optical densities of the surface blots divided by the input fraction standardized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control relative to the WT signal). The data are provided as the mean ± SEM (* p < 0.05).