Disruption of asparagine-linked glycosylation to rescue and alter gating of the Na V 1.5-Na + channel

SCN5A gene encodes the voltage-gated sodium channel Na 1.5 which is composed of a pore-forming α subunit of the channel. Asparagine (N)-linked glycosylation is one of the common post-translational modifications in proteins. The aim of this study was to investigate impact of N-linked glycosylation disruption on the Na channel, and the mechanism by which glycosylation regulates the current density and gating properties of the Na channel. The Na 1.5-Na channel isoform (α submit) derived from human was stably expressed in human embryonic kidney (HEK)-293 cells (Nav1.5-HEK cell). We applied the whole-cell patch-clamp technique to study the impact of N-linked glycosylation disruption in Nav1.5-HEK cell. Inhibition of the N-glycosylation with tunicamycin caused a significant increase of Na 1.5 channel current (I ) when applied for 24 h. Tunicamycin shifted the steady-state inactivation curve to the hyperpolarization direction, whereas the activation curve was unaffected. Recovery from inactivation was prolonged, while the fast phase (τ ) and the slow phase (τ ) of the current decay was unaffected by tunicamycin. I was unaffected by tunicamycin in the present of a proteasome inhibitor MG132 [N-[(phenylmethoxy)carbonyl]-L-leucy-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide], while it was significantly increased by tunicamycin in the presence of a lysosome inhibitor butyl methacrylate (BMA). These findings suggest that N-glycosylation disruption rescues the Na 1.5 channel possibly through the alteration of ubiquitin-proteasome activity, and changes gating properties of the Na 1.5 channel by modulating glycan milieu of the channel protein.