Disruption of asparagine-linked glycosylation to rescue and alter gating of the NaV1.5-Na+ channel.

Pu Wang, Xiufang Zhu, Mengyan Wei, Yangong Liu, Kenshi Yoshimura, Mingqi Zheng, Gang Liu, Shinichiro Kume, Tatsuki Kurokawa, Katsushige Ono

Heart and vessels 2021 Apr

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SCN5A gene encodes the voltage-gated sodium channel NaV1.5 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 NaV1.5-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 NaV1.5 channel current (INa) 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 (τfast) and the slow phase (τslow) of the current decay was unaffected by tunicamycin. INa 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 NaV1.5 channel possibly through the alteration of ubiquitin-proteasome activity, and changes gating properties of the NaV1.5 channel by modulating glycan milieu of the channel protein.

Citation

Pu Wang, Xiufang Zhu, Mengyan Wei, Yangong Liu, Kenshi Yoshimura, Mingqi Zheng, Gang Liu, Shinichiro Kume, Tatsuki Kurokawa, Katsushige Ono. Disruption of asparagine-linked glycosylation to rescue and alter gating of the NaV1.5-Na+ channel. Heart and vessels. 2021 Apr;36(4):589-596