N-linked glycosylation sites determine HERG channel surface membrane expression

1. Long QT syndrome (LQT) is an electrophysiological disorder that can lead to sudden death from cardiac arrhythmias. One form of LQT has been attribu ted to mutations in the human ether-a-go-go-related gene (HERG) that encode s 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 e xtracellular 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 glycosyla tion to the surface localization of HERG channels. 2. Tunicamycin, an inhibitor of N-linked glycosylation, blocked normal surf ace membrane expression of a HERG-green fluorescent protein (GFP) fusion pr otein (HERG(GFP)) transiently expressed in human embryronic 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 presenc e of N-linked carbohydrate moieties. Mutations. at each of the two putative extracellular N-linked glycosylation sites (N598Q and N629Q) led to a peri nuclear subcellular localization of HERG(GFP) stably expressed in HEK 293 c ells, with no surface membrane expression. Furthermore, patch clamp analysi s revealed that there was a virtual absence of HERG current in the N-glycos ylation mutants. 4. Taken together, these results strongly suggest that N-linked glycosylati on is required for surface membrane expression of HERG. These findings may provide insight into a mechanism responsible for LQT2 due to N-linked glyco sylation-related mutations of HERG.