title: N-Linked glycan site occupancy impacts the distribution of a potassium channel in the cell body and outgrowths of neuronal-derived cells.
abstract: BACKGROUND
  Vacancy of occupied N-glycosylation sites of glycoproteins is quite disruptive to a multicellular organism, as underlined by congenital disorders of glycosylation. Since a neuronal component is typically associated with this disease, we evaluated the impact of N-glycosylation processing of a neuronal voltage gated potassium channel, Kv3.1b, expressed in a neuronal-derived cell line, B35 neuroblastoma cells.
  
  
  METHODS
  Total internal reflection fluorescence and differential interference contrast microscopy measurements of live B35 cells expressing wild type and glycosylation mutant Kv3.1b proteins were used to evaluate the distribution of the various forms of the Kv3.1b protein in the cell body and outgrowths. Cell adhesion assays were also employed.
  
  
  RESULTS
  Microscopy images revealed that occupancy of both N-glycosylation sites of Kv3.1b had relatively similar amounts of Kv3.1b in the outgrowth and cell body while vacancy of one or both sites led to increased accumulation of Kv3.1b in the cell body. Further both the fully glycosylated and partially glycosylated N229Q Kv3.1b proteins formed higher density particles in outgrowths compared to cell body. Cellular assays demonstrated that the distinct spatial arrangements altered cell adhesion properties.
  
  
  CONCLUSIONS
  Our findings provide direct evidence that occupancy of the N-glycosylation sites of Kv3.1b contributes significantly to its lateral heterogeneity in membranes of neuronal-derived cells, and in turn alters cellular properties.
  
  
  CONCLUSIONS
  Our study demonstrates that N-glycans of Kv3.1b contain information regarding the association, clustering, and distribution of Kv3.1b in the cell membrane, and furthermore that decreased occupancy caused by congenital disorders of glycosylation may alter the biological activity of Kv3.1b.
authors.affiliation: Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, NC 27834, USA.
authors.forename: M K
authors.initials: MK
authors.lastname: Hall
authors.forename: D A
authors.initials: DA
authors.lastname: Weidner
authors.forename: C J
authors.initials: CJ
authors.lastname: Bernetski
authors.forename: R A
authors.initials: RA
authors.lastname: Schwalbe
mesh_terms: D015153:Blotting, Western; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism*; D049109:Cell Proliferation; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D051670:Shaw Potassium Channels / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions; D014407:Tumor Cells, Cultured
chemical_list: C489597:KCNC1 protein, human; D011134:Polysaccharides; D051670:Shaw Potassium Channels; D006026:Glycoside Hydrolases
keywords: CB; CDG; DIC; Excitable membrane; High density potassium channel cluster; Kv channel; Membrane glycoprotein; Membrane trafficking; N-glycosylation; Neu; OG; Potassium channel; TIRF; cell body; congenital disorder of glycosylation; differential interference contrast; neuraminidase; outgrowth; total internal reflection fluorescence; voltage-gating potassium channel