Role of glycosylation in cell surface expression and stability of HERG potassium channels.
The human ether-à-go-go-related gene (HERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel in the heart. We previously showed that HERG channel protein is modified by N-linked glycosylation. HERG protein sequence contains two extracellular consensus sites for N-linked glycosylation (N598, N629). In this study, we used the approaches of site-directed mutagenesis and biochemical modification to inhibit N-linked glycosylation and studied the role of glycosylation in the cell surface expression and turnover of HERG channels. Our results show that N598 is the only site for N-linked glycosylation and that glycosylation is not required for the cell surface expression of functional HERG channels. In contrast, N629 is not used for glycosylation, but mutation of this site (N629Q) causes a protein trafficking defect, which results in its intracellular retention. Pulse-chase experiments show that the turnover rate of nonglycosylated HERG channel is faster than that of the glycosylated form, suggesting that N-linked glycosylation plays an important role in HERG channel stability.
Gong
Qiuming
Q
Division of Molecular Medicine, Department of Medicine, Oregon Health and Science University, Portland, Oregon 97201, USA.
Anderson
Corey L
CL
January
Craig T
CT
Zhou
Zhengfeng
Z
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D015153:Blotting, Western; D027682:Cation Transport Proteins; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004268:DNA-Binding Proteins; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; D006031:Glycosylation / Q000187:drug effects; D049452:Green Fluorescent Proteins; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D027682:Cation Transport Proteins; D004268:DNA-Binding Proteins; C072312:ERG protein, human; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; C092509:KCNH6 protein, human; D008164:Luminescent Proteins; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG; D014415:Tunicamycin; D049452:Green Fluorescent Proteins