Potassium channel alpha and beta subunits assemble in the endoplasmic reticulum.
We have characterized the maturation of Shaker K+ channel protein and the cellular site of assembly of pore-forming alpha and cytoplasmic beta subunits in a transfected mammalian cell line. Shaker protein is made as a partially glycosylated, immature precursor that is converted to a fully glycosylated, mature product. Shaker protein did not mature when transport from the endoplasmic reticulum (ER) to the Golgi apparatus was blocked. Consistent with this finding, only the immature form was sensitive to digestion with endoglycosidase H. These results indicate that the immature protein is core-glycosylated in the ER, whereas the oligosaccharides of the mature protein have been further processed in the Golgi compartment. After inhibiting ER-to-Golgi transport, the oligomeric state of Shaker subunits was assessed by cross-linking in intact cells or by solubilization and sucrose gradient sedimentation. The results indicate that Shaker subunits assemble with each other in the ER. When co-expressed, the Kvbeta2 subunit also associated with Shaker in the ER. Assembly with the beta2 subunit did not increase the rate or extent of Shaker protein maturation. Our results indicate that the biogenesis of Shaker K+ channels in vivo involves core glycosylation and subunit assembly in the ER, followed by efficient transfer to the Golgi apparatus where the oligosaccharides are modified.
Nagaya
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Department of Physiology, School of Medicine, and Molecular Biology Institute, University of California, Los Angeles, California 90095-1751, USA.
Papazian
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DM
D000818:Animals; D020126:Brefeldin A; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D003080:Cold Temperature; D003517:Cyclopentanes / Q000494:pharmacology; D004220:Disulfides; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007668:Kidney; D046911:Macromolecular Substances; D008322:Mammals; D009844:Oligosaccharides / Q000096:biosynthesis; D015221:Potassium Channels / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D051637:Shaker Superfamily of Potassium Channels; D014162:Transfection
D003517:Cyclopentanes; D004220:Disulfides; D046911:Macromolecular Substances; D009844:Oligosaccharides; D015221:Potassium Channels; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D051637:Shaker Superfamily of Potassium Channels; D020126:Brefeldin A