title: Apical sorting of a voltage- and Ca2+-activated K+ channel alpha -subunit in Madin-Darby canine kidney cells is independent of N-glycosylation.
abstract: The voltage- and Ca(2+)-activated K(+) (K(V,Ca)) channel is expressed in a variety of polarized epithelial cells seemingly displaying a tissue-dependent apical-to-basolateral regionalization, as revealed by electrophysiology. Using domain-specific biotinylation and immunofluorescence we show that the human channel K(V,Ca) alpha-subunit (human Slowpoke channel, hSlo) is predominantly found in the apical plasma membrane domain of permanently transfected Madin-Darby canine kidney cells. Both the wild-type and a mutant hSlo protein lacking its only potential N-glycosylation site were efficiently transported to the cell surface and concentrated in the apical domain even when they were overexpressed to levels 200- to 300-fold higher than the density of intrinsic Slo channels. Furthermore, tunicamycin treatment did not prevent apical segregation of hSlo, indicating that endogenous glycosylated proteins (e.g., K(V,Ca) beta-subunits) were not required. hSlo seems to display properties for lipid-raft targeting, as judged by its buoyant distribution in sucrose gradients after extraction with either detergent or sodium carbonate. The evidence indicates that the hSlo protein possesses intrinsic information for transport to the apical cell surface through a mechanism that may involve association with lipid rafts and that is independent of glycosylation of the channel itself or an associated protein. Thus, this particular polytopic model protein shows that glycosylation-independent apical pathways exist for endogenous membrane proteins in Madin-Darby canine kidney cells.
authors.affiliation: Departamento de Inmunologia Clinica y Reumatologia, Facultad de Medicina, Departamento de Biologia Celular y Molecular, Pontificia Universidad Católica de Chile, Santiago.
authors.forename: M
authors.initials: M
authors.lastname: Bravo-Zehnder
authors.forename: P
authors.initials: P
authors.lastname: Orio
authors.forename: A
authors.initials: A
authors.lastname: Norambuena
authors.forename: M
authors.initials: M
authors.lastname: Wallner
authors.forename: P
authors.initials: P
authors.lastname: Meera
authors.forename: L
authors.initials: L
authors.lastname: Toro
authors.forename: R
authors.initials: R
authors.lastname: Latorre
authors.forename: A
authors.initials: A
authors.lastname: González
mesh_terms: D000818:Animals; D002118:Calcium / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D002499:Centrifugation, Density Gradient; D004285:Dogs; D006031:Glycosylation; D006801:Humans; D007668:Kidney; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008564:Membrane Potentials / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D024681:Potassium Channels, Calcium-Activated; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
chemical_list: D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D021122:Protein Subunits; D011994:Recombinant Proteins; D002118:Calcium