Effects of N-glycosylation on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels

Biochem J. 2015 Feb 15;466(1):77-84. doi: 10.1042/BJ20140692.

Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarization and conduct an inward cation current, which contributes to rhythmic electrical activity of neural and cardiac pacemaker cells. HCN channels have been shown to undergo N-linked glycosylation, and the N-glycosylation has been shown to be required for membrane trafficking and possibly function. In this study, recombinant wild-type (WT) and glycosylation-defective N380Q HCN2 channels were individually or co-expressed in HEK-293 cells. We demonstrate that glycosylation is required for trafficking to the plasma membrane and for the stability of HCN channels in the cell. Interestingly, the heteromeric HCN2 channels of WT and glycosylation-defective N380Q have been observed on cell membranes, indicating that not all four subunits of a tetrameric HCN2 channel need to be glycosylated for HCN2 channels to traffic to plasma membranes. Subsequently, we investigate the effect of N-glycosylation on the function of HCN2 channels. We developed a fluorescence-based flux assay, which makes it possible to establish a negative potential inside liposomes to open HCN2 channels. Using this flux assay, we demonstrate that glycosylation-defective N380Q HCN2 channels reconstituted into liposomes function similarly to WT HCN2 channels. This suggests that N-glycosylation is not required for HCN2 channels to function.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Membrane / chemistry
  • Cell Membrane / metabolism*
  • Gene Expression
  • Glycosylation
  • HEK293 Cells
  • Humans
  • Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels / genetics
  • Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels / metabolism*
  • Models, Biological
  • Mutation
  • Potassium Channels / genetics
  • Potassium Channels / metabolism*
  • Protein Multimerization
  • Protein Subunits / genetics
  • Protein Subunits / metabolism*
  • Protein Transport
  • Proteolipids / chemistry
  • Proteolipids / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism

Substances

  • HCN2 protein, human
  • Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels
  • Potassium Channels
  • Protein Subunits
  • Proteolipids
  • Recombinant Proteins
  • proteoliposomes