Disruption of asparagine-linked glycosylation to rescue and alter gating of the Na_V1.5-Na⁺ channel

SCN5A gene encodes the voltage-gated sodium channel Na_V1.5 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_V1.5-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_V1.5 channel current (I_Na) 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_fast) and the slow phase (τ_slow) of the current decay was unaffected by tunicamycin. I_Na 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_V1.5 channel possibly through the alteration of ubiquitin–proteasome activity, and changes gating properties of the Na_V1.5 channel by modulating glycan milieu of the channel protein.