Allowed N-glycosylation sites on the Kv1.2 potassium channel S1-S2 linker: Implications for linker secondary structure and the glycosylation effect on channel function

Jing Zhu, Itaru Watanabe, Amanda Poholek, Matthew Koss, Barbara Gomez, Chaowen Yan, Esperanza Recio-Pinto, William B. Thornhill

Research output: Contribution to journalArticlepeer-review

29 Scopus citations

Abstract

N-glycosylation is a post-translational modification that plays a role in the trafficking and/or function of some membrane proteins. We have shown previously that N-glycosylation affected the function of some Kv1 voltage-gated potassium (K+) channels [Watanabe, Wang, Sutachan, Zhu, Recio-Pinto and Thornhill (2003) J. Physiol. (Cambridge, U.K.) 550, 51-66]. Kv1 channel S1-S2 linkers vary in length but their N-glycosylation sites are at similar relative positions from the S1 or S2 membrane domains. In the present study, by a scanning mutagenesis approach, we determined the allowed N-glycosylation sites on the Kv1.2S1-S2 linker, which has 39 amino acids, by engineering N-glycosylation sites and assaying for glycosylation, using their sensitivity to glycosidases. The middle section of the linker (54% of linker) was glycosylated at every position, whereas both end sections (46% of linker) near the S1 or S2 membrane domains were not. These findings suggested that the middle section of the S1-S2 linker was accessible to the endoplasmic reticulum glycotransferase at every position and was in the extracellular aqueous phase, and presumably in a flexible conformation. We speculate that the S1-S2 linker is mostly a coiled-loop structure and that the strict relative position of native glycosylation sites on these linkers may be involved in the mechanism underlying the functional effects of glycosylation on some Kv1K+ channels. The S3-S4 linker, with 16 amino acids and no N-glycosylation site, was not glycosylated when an N-glycosylation site was added. However, an extended linker, with an added N-linked site, was glycosylated, which suggested that the native linker was not glycosylated due to its short length. Thus other ion channels or membrane proteins may also have a high glycosylation potential on a linker but yet have similarly positioned native N-glycosylation sites among isoforms. This may imply that the native position of the N-glycosylation site may be important if the carbohydrate tree plays a role in the folding, stability, trafficking and/or function of the protein.

Original languageEnglish (US)
Pages (from-to)769-775
Number of pages7
JournalBiochemical Journal
Volume375
Issue number3
DOIs
StatePublished - Nov 1 2003
Externally publishedYes

Keywords

  • Endoplasmic reticulum
  • Extracellular linker
  • N-glycosylation
  • Voltage-gated potassium channel

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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