Apical sorting of a voltage- and Ca2+-activated K+ channel alpha-subunit in Madin-Darby canine kidney cells is independent of N-glycosylation
The voltage- and Ca2+-acttiated K+ (K-V,K-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,K-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,K-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.
|Título según WOS:||Apical sorting of a voltage- and Ca2+-activated K+ channel alpha-subunit in Madin-Darby canine kidney cells is independent of N-glycosylation|
|Título según SCOPUS:||Apical sorting of a voltage- and Ca2+-activated K+ channel ?-subunit in Madin-Darby canine kidney cells is independent of N-glycosylation|
|Título de la Revista:||PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA|
|Editorial:||NATL ACAD SCIENCES|
|Fecha de publicación:||2000|
|Página de inicio:||13114|