title: N-glycosylation promotes the cell surface expression of Kv1.3 potassium channels.
abstract: The voltage-gated potassium channel Kv1.3 plays an essential role in modulating membrane excitability in many cell types. Kv1.3 is a heavily glycosylated membrane protein. Two successive N-glycosylation consensus sites, N228NS and N229ST, are present on the S1-S2 linker of rat Kv1.3. Our data suggest that Kv1.3 contains only one N-glycan and it is predominantly attached to N229 in the S1-S2 extracellular linker. Preventing N-glycosylation of Kv1.3 significantly decreased its surface protein level and surface conductance density level, which were ∼ 49% and ∼ 46% respectively of the level of wild type. Supplementation of N-acetylglucosamine (GlcNAc), l-fucose or N-acetylneuraminic acid to the culture medium promoted Kv1.3 surface protein expression, whereas supplementation of d-glucose, d-mannose or d-galactose did not. Among the three effective monosaccharides/derivatives, adding GlcNAc appeared to reduce sialic acid content and increase the degree of branching in the N-glycan of Kv1.3, suggesting that the N-glycan structure and composition had changed. Furthermore, the cell surface half-life of the Kv1.3 surface protein was increased upon GlcNAc supplementation, indicating that it had decreased internalization. The GlcNAc effect appears to apply mainly to membrane proteins containing complex type N-glycans. Thus, N-glycosylation promotes Kv1.3 cell surface expression; supplementation of GlcNAc increased Kv1.3 surface protein level and decreased its internalization, presumably by a combined effect of decreased branch size and increased branching of the N-glycan.
authors.affiliation: Department of Biological Sciences and Center for Cancer, Genetic Diseases and Gene Regulation, Fordham University, Bronx, NY, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhu
authors.forename: Jenny
authors.initials: J
authors.lastname: Yan
authors.forename: William B
authors.initials: WB
authors.lastname: Thornhill
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D051664:Kv1.3 Potassium Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C496975:Kcna3 protein, rat; D051664:Kv1.3 Potassium Channel; D011134:Polysaccharides; D011994:Recombinant Proteins; D000117:Acetylglucosamine