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

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Abstract

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.

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Abbreviations

N-linked:

Asparagine-linked

HEK cells:

Human embryonic kidney

MG132:

N-[(phenylmethoxy)carbonyl]-l- leucy-N-[(1S)-1-formyl-3-methylbutyl]- l-leucinamide

BMA:

Butyl methacrylate

DMEM:

Dulbecco's modified Eagle's medium

INa :

Voltage-gated sodium channel current

LQT3:

Long QT syndrome type 3

TEA-Cl:

Tetraethylammonium chloride

TEA-OH:

Tetraethylammonium hydroxide

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

G:

Conductance

I-V curve:

Current–voltage curve

ER:

Endoplasmic reticulum

FOXO1:

Forkhead box protein O1

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Funding

This work was supported in part by JSPS KAKEN 25460292 (KO) from the Japan Society for the Promotion of Science, Tokyo.

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Author notes

  1. Pu Wang, Xiufang Zhu and Mengyan Wei contributed equally to this work.

Authors and Affiliations

  1. Department of Cardiology, The First Hospital of Hebei Medical University, 89 Donggang Road, Shijiazhuang, Hebei Province, 050031, People’s Republic of China

    Pu Wang, Xiufang Zhu, Mengyan Wei, Yangong Liu, Mingqi Zheng & Gang Liu

  2. Department of Pathophysiology, Oita University School of Medicine, Yufu, Oita, 879-5593, Japan

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

Authors
  1. Pu Wang
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  2. Xiufang Zhu
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  3. Mengyan Wei
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  4. Yangong Liu
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Contributions

All authors gave their informed consent prior to their inclusion in the study. PW, MW, XZ and YL conceived and planned the experiments. PW, MW, and XZ conducted the experiments. KY, MG and, GL contributed to data analysis and interpretation. SK, TK and KO took the lead in writing the manuscript.

Corresponding author

Correspondence to Katsushige Ono.

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Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethics approval

The experiments conducted in this work were approved by approved in advance by the Ethics Review Committee for Animal Experimentation of Oita University School of Medicine, and in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996).

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Wang, P., Zhu, X., Wei, M. et al. Disruption of asparagine-linked glycosylation to rescue and alter gating of the NaV1.5-Na+ channel. Heart Vessels 36, 589–596 (2021). https://doi.org/10.1007/s00380-020-01736-4

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  • DOI: https://doi.org/10.1007/s00380-020-01736-4

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