Disruption of asparagine-linked glycosylation to rescue and alter gating of the NaV1.5-Na+ channel
Disruption of asparagine-linked glycosylation to rescue and alter gating of the NaV1.5-Na+ channel
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DOI：
10.1007/s00380-020-01736-4
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发表时间：
2021-01
影响因子：
1.5
通讯作者：
Pu Wang;Xiufang Zhu;Mengyan Wei;Yangong Liu;Kenshi Yoshimura;Mingqi Zheng;Gang Liu;S. Kume;Tatsuki Kurokawa;K. Ono
中科院分区：
文献类型：
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作者：
Pu Wang;Xiufang Zhu;Mengyan Wei;Yangong Liu;Kenshi Yoshimura;Mingqi Zheng;Gang Liu;S. Kume;Tatsuki Kurokawa;K. Ono
SCN 5A基因编码电压门控钠通道NaV 1。5的α亚基组成的通道。天冬酰胺（N）连接的糖基化是蛋白质中常见的翻译后修饰之一。本研究的目的是研究N-连接糖基化破坏对Na+通道的影响，以及糖基化调节Na+通道电流密度和门控特性的机制。NAV1。来源于人的5-Na+通道亚型（α submit）在人胚肾（HEK）-293细胞（Nav 1. 5-HEK细胞）。我们应用全细胞膜片钳技术来研究Nav 1中N-连接糖基化破坏的影响。5-HEK细胞。用衣霉素抑制N-糖基化引起NaV 1的显著增加。施加24 h时的5通道电流（INa）。衣霉素使稳态失活曲线向超极化方向移动，而激活曲线不受影响。失活的恢复时间延长，而电流衰减的快相（τfast）和慢相（τslow）不受衣霉素的影响。在蛋白酶体抑制剂MG 132 [N-[（苯甲氧基）羰基]-L-亮氨酰基-N-[（1 S）-1-甲酰基-3-甲基丁基]-L-亮氨酰胺]存在下，衣霉素不影响INa，而在溶酶体抑制剂甲基丙烯酸丁酯（BMA）存在下，衣霉素显著增加INa。这些发现表明N-糖基化破坏拯救了NaV 1。5通道可能通过改变泛素-蛋白酶体活性，并改变NaV 1的门控特性。通过调节通道蛋白的聚糖环境来调节5通道。
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.