Characterization of N-glycosylation consensus sequences in the Kv3.1 channel.
N-Glycosylation is a cotranslational and post-translational process of proteins that may influence protein folding, maturation, stability, trafficking, and consequently cell surface expression of functional channels. Here we have characterized two consensus N-glycosylation sequences of a voltage-gated K+ channel (Kv3.1). Glycosylation of Kv3.1 protein from rat brain and infected Sf9 cells was demonstrated by an electrophoretic mobility shift assay. Digestion of total brain membranes with peptide N glycosidase F (PNGase F) produced a much faster-migrating Kv3.1 immunoband than that of undigested brain membranes. To demonstrate N-glycosylation of wild-type Kv3.1 in Sf9 cells, cells were treated with tunicamycin. Also, partially purified proteins were digested with either PNGase F or endoglycosidase H. Attachment of simple-type oligosaccharides at positions 220 and 229 was directly shown by single (N229Q and N220Q) and double (N220Q/N229Q) Kv3.1 mutants. Functional measurements and membrane fractionation of infected Sf9 cells showed that unglycosylated Kv3.1s were transported to the plasma membrane. Unitary conductance of N220Q/N229Q was similar to that of the wild-type Kv3.1. However, whole cell currents of N220Q/N229Q channels had slower activation rates, and a slight positive shift in voltage dependence compared to wild-type Kv3.1. The voltage dependence of channel activation for N229Q and N220Q was much like that for N220Q/N229Q. These results demonstrate that the S1-S2 linker is topologically extracellular, and that N-glycosylation influences the opening of the voltage-dependent gate of Kv3.1. We suggest that occupancy of the sites is critical for folding and maturation of the functional Kv3.1 at the cell surface.
Brooks
Natasha L
NL
Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA.
Corey
Melissa J
MJ
Schwalbe
Ruth A
RA
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001921:Brain / Q000166:cytology; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D016384:Consensus Sequence; D017124:Conserved Sequence; D006031:Glycosylation; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000494:pharmacology; D051381:Rats; D017386:Sequence Homology, Amino Acid; D051670:Shaw Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000166:cytology / Q000187:drug effects; D014415:Tunicamycin / Q000494:pharmacology
D008565:Membrane Proteins; D051670:Shaw Potassium Channels; D014415:Tunicamycin; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase