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N‐linked glycosylation sites determine HERG channel surface membrane expression
Author(s) -
Petrecca Kevin,
Atanasiu Roxana,
Akhavan Armin,
Shrier Alvin
Publication year - 1999
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.1999.041ad.x
Subject(s) - herg , hek 293 cells , glycosylation , microbiology and biotechnology , n linked glycosylation , chemistry , green fluorescent protein , patch clamp , biology , biochemistry , potassium channel , biophysics , gene , receptor , glycoprotein , glycan
1 Long QT syndrome (LQT) is an electrophysiological disorder that can lead to sudden death from cardiac arrhythmias. One form of LQT has been attributed to mutations in the human ether‐a‐go‐go‐ related gene ( HERG ) that encodes a voltage‐gated cardiac K + channel. While a recent report indicates that LQT in some patients is associated with a mutation of HERG at a consensus extracellular N‐linked glycosylation site (N629), earlier studies failed to identify a role for N‐linked glycosylation in the functional expression of voltage‐gated K + channels. In this study we used pharmacological agents and site‐directed mutagenesis to assess the contribution of N‐linked glycosylation to the surface localization of HERG channels. 2 Tunicamycin, an inhibitor of N‐linked glycosylation, blocked normal surface membrane expression of a HERG‐green fluorescent protein (GFP) fusion protein (HERG GFP ) transiently expressed in human embryonic kidney (HEK 293) cells imaged with confocal microscopy. 3 Immunoblot analysis revealed that N‐glycosidase F shifted the molecular mass of HERG GFP , stably expressed in HEK 293 cells, indicating the presence of N‐linked carbohydrate moieties. Mutations at each of the two putative extracellular N‐linked glycosylation sites (N598Q and N629Q) led to a perinuclear subcellular localization of HERG GFP stably expressed in HEK 293 cells, with no surface membrane expression. Furthermore, patch clamp analysis revealed that there was a virtual absence of HERG current in the N‐glycosylation mutants. 4 Taken together, these results strongly suggest that N‐linked glycosylation is required for surface membrane expression of HERG. These findings may provide insight into a mechanism responsible for LQT2 due to N‐linked glycosylation‐related mutations of HERG .