Glycosylation Increases Potassium Channel Stability and Surface Expression in Mammalian Cells

Rajesh Khanna, Michael P. Myers, Muriel Lainé, Diane M. Papazian

Research output: Contribution to journalArticle

70 Scopus citations

Abstract

N-linked glycosylation is not required for the cell surface expression of functional Shaker potassium channels in Xenopus oocytes (Santacruz-Toloza, L., Huang, Y., John, S. A., and Papazian, D. M. (1994) Biochemistry 33, 5607-5613). We have now investigated whether glycosylation increases the stability, cell surface expression, and proper folding of Shaker protein expressed in mammalian cells. The turnover rates of wild-type protein and an unglycosylated mutant (N259Q,N263Q) were compared in pulse-chase experiments. The wild-type protein was stable, showing little degradation after 48 h. In contrast, the unglycosylated mutant was rapidly degraded (t1/2 = ∼18 h). Lactacystin slowed the degradation of the mutant protein, implicating cytoplasmic proteasomes in its turnover. Rapid lactacystin-sensitive degradation could be conferred on wild-type Shaker by a glycosylation inhibitor. Expression of the unglycosylated mutant on the cell surface, assessed using immunofluorescence microscopy and biotinylation, was dramatically reduced compared with wild type. Folding and assembly were analyzed by oxidizing intersubunit disulfide bonds, which provides a fortuitous hallmark of the native structure. Surprisingly, formation of disulfidebonded adducts was quantitatively similar in the wild-type and unglycosylated mutant proteins. Our results indicate that glycosylation increases the stability and cell surface expression of Shaker protein but has little effect on acquisition of the native structure.

Original languageEnglish (US)
Pages (from-to)34028-34034
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number36
DOIs
StatePublished - Sep 7 2001
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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