Glycosylation affects the rate of traffic of the Shaker potassium channel through the secretory pathway.
We have examined the effect of glycosylation on the traffic of the voltage-gated Shaker potassium channel through the secretory pathway of mammalian cells. Shaker is glycosylated on two asparagines (N259 and N263) in the first extracellular loop. Electrophysiological experiments indicate that glycosylation is not necessary for channel integrity [Santacruz-Toloza et al. (1994) Biochemistry 33, 5607]. Consistent with this, we observe that unglycosylated N259Q+N263Q mutant channel forms oligomers as efficiently as the wild type and that this occurs in the endoplasmic reticulum. We have compared the kinetics of secretory traffic of the wild-type glycosylated and the N259Q+N263Q unglycosylated channels. Surface biotinylation of newly synthesized proteins indicates that the rate of delivery of the unglycosylated channel to the cell surface is slower than that of wild type. We have further dissected channel traffic using quantitative imaging. We observe that mutant channel traffics more slowly from the endoplasmic reticulum to the Golgi than wild type at 20 degrees C. This may contribute to the slowed delivery of the mutant to the cell surface. Neither the surface fraction at steady state nor the stability of Shaker is significantly affected by glycosylation in COS cells.
de Souza
Natalie F
NF
Laboratory of Cellular Biophysics, The Rockefeller University, New York, New York 10021, USA.
Simon
Sanford M
SM
D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D019921:Biotinylation; D019556:COS Cells; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum; D006031:Glycosylation; D006056:Golgi Apparatus / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D015640:Ion Channel Gating; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D051637:Shaker Superfamily of Potassium Channels; D014162:Transfection
D015221:Potassium Channels; D011994:Recombinant Proteins; D051637:Shaker Superfamily of Potassium Channels; D001216:Asparagine