THE ROLE OF GLYCOSYLATION IN CARDIAC REPOLARIZATION

Abstract

The human ether-a-go-go-related gene (hERG) encodes the pore-forming alpha subunit of the rapidly activating delayed rectifier potassium current (IKr), which is important for cardiac repolarization. Dysfunction of hERG due to drug block or mutations can result in reduced IKr, which is associated with the development of Long QT Syndrome (LQTS). LQTS is a cardiac arrhythmia seen on an ECG as an increased QT interval, and can result in syncope or sudden death. hERG has 6 transmembrane domains, ranging from S1 to S6, and a complex N-linked oligosaccharide chain which resides on the extracellular S5 pore linker region. Glycosylation chains have been associated with numerous roles in cell and protein function, such as protection from protease-induced degradation. The role of the glycosylation chain found on hERG in the channel’s function and expression has not yet been fully revealed, and could provide insight into the cellular mechanisms behind hERG-related LQTS. In the current study, we aim to investigate the relationship between hERG susceptibility to protease degradation and the presence of the glycosylation chain. Human Embryonic Kidney Cells expressing hERG channels were cultured with the glycosylation-reducing agents tunicamycin (an antibiotic that prevents the addition of N-linked glycosylation) and neuraminidase (an exoglycosidase that removes sialic acid residues). Tunicamycin and neuraminidase-treated cells were then subjected to protease degradation by Proteinase K (PK) and Calpain-1. hERG protease susceptibility was measured either through Western blot fragment intensity quantification or through whole cell voltage clamp analysis. Scorpion toxin BeKm-1 was added to cell culture media with PK, then washed out prior to protease susceptibility analysis. We demonstrate that unglycosylated hERG channels are more susceptible to protease-induced degradation by PK and Calpain-1. Removal of the end-chain sialic acid residues using the enzyme neuraminidase did not alter the glycosylation-mediated protease protection afforded to wild-type hERG channels. Additionally, we showed that the scorpion toxin BeKm-1 requires the glycosylation chain to protect hERG channels from PK cut. Our findings indicate that the glycosylation chain found on mature hERG channels plays a key role in protecting the channel from protease digestion, and suggests a glycosylation-mediated mechanism for the BeKm-1-mediated hERG channel protection from PK cut.