title: N-glycosylation-dependent control of functional expression of background potassium channels K2P3.1 and K2P9.1.
abstract: Two-pore domain potassium (K(2P)) channels play fundamental roles in cellular processes by enabling a constitutive leak of potassium from cells in which they are expressed, thus influencing cellular membrane potential and activity. Hence, regulation of these channels is of critical importance to cellular function. A key regulatory mechanism of K(2P) channels is the control of their cell surface expression. Membrane protein delivery to and retrieval from the cell surface is controlled by their passage through the secretory and endocytic pathways, and post-translational modifications regulate their progression through these pathways. All but one of the K(2P) channels possess consensus N-linked glycosylation sites, and here we demonstrate that the conserved putative N-glycosylation site in K(2P)3.1 and K(2P)9.1 is a glycan acceptor site. Patch clamp analysis revealed that disruption of channel glycosylation reduced K(2P)3.1 current, and flow cytometry was instrumental in attributing this to a decreased number of channels on the cell surface. Similar findings were observed when cells were cultured in reduced glucose concentrations. Disruption of N-linked glycosylation has less of an effect on K(2P)9.1, with a small reduction in number of channels on the surface observed, but no functional implications detected. Because nonglycosylated channels appear to pass through the secretory pathway in a manner comparable with glycosylated channels, the evidence presented here suggests that the decreased number of nonglycosylated K(2P)3.1 channels on the cell surface may be due to their decreased stability.
authors.affiliation: Human Development and Health, Centre for Human Development, Stem Cells and Regeneration, Faculty of Medicine, University of Southampton, Southampton SO16 6YD, United Kingdom.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Mant
authors.forename: Sarah
authors.initials: S
authors.lastname: Williams
authors.forename: Laura
authors.initials: L
authors.lastname: Roncoroni
authors.forename: Eleanor
authors.initials: E
authors.lastname: Lowry
authors.forename: Daniel
authors.initials: D
authors.lastname: Johnson
authors.forename: Ita
authors.initials: I
authors.lastname: O'Kelly
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002522:Chlorocebus aethiops; D005947:Glucose / Q000494:pharmacology; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009419:Nerve Tissue Proteins; D024683:Potassium Channels, Tandem Pore Domain / Q000737:chemistry / Q000378:metabolism*; D055550:Protein Stability / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D016415:Sequence Alignment; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism
chemical_list: D006023:Glycoproteins; C407082:Kcnk9 protein, rat; D050505:Mutant Proteins; D009419:Nerve Tissue Proteins; D024683:Potassium Channels, Tandem Pore Domain; C420132:potassium channel subfamily K member 3; D005947:Glucose