N-linked glycosylation determines cell surface expression of two-pore-domain K+ channel TRESK.
Within the first external loop of mouse and human TRESK subunits one or two N-glycosylation consensus sites were identified, respectively. Using site directed mutagenesis and Western immunoblotting a single residue of both orthologues was found to be glycosylated upon heterologous expression. Two-electrode voltage-clamp recordings from Xenopus oocytes revealed that current amplitudes of N-glycosylation mutants were reduced by 80% as compared to wildtype TRESK. To investigate membrane targeting, GFP-tagged TRESK subunits were expressed in Xenopus oocytes and fluorescence intensity at the cell surface was measured by confocal microscopy. Signals of the N-glycosylation mutants were reduced by >50%, indicating that their lower current amplitudes substantially result from inadequate surface expression of the channel.
Egenberger
Brigitte
B
Institute of Physiology, Department of Neurophysiology, University of Würzburg, 97070 Würzburg, Germany.
Polleichtner
Georg
G
Wischmeyer
Erhard
E
Döring
Frank
F
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
D015221:Potassium Channels; C476574:KCNK18 protein, human; C486498:Kcnk18 protein, mouse