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Disruption of protein quality control of the human ether-à-go-go related gene K+ channel results in profound long QT syndrome
Heart Rhythm ( IF 5.5 ) Pub Date : 2021-10-09 , DOI: 10.1016/j.hrthm.2021.10.005
Hannah A Ledford 1 , Lu Ren 1 , Phung N Thai 1 , Seojin Park 2 , Valeriy Timofeyev 1 , Padmini Sirish 3 , Wilson Xu 1 , Aiyana M Emigh 4 , James R Priest 5 , Marco V Perez 5 , Euan A Ashley 5 , Vladimir Yarov-Yarovoy 4 , Ebenezer N Yamoah 6 , Xiao-Dong Zhang 3 , Nipavan Chiamvimonvat 3
Affiliation  

Background

Long QT syndrome (LQTS) is a hereditary disease that predisposes patients to life-threatening cardiac arrhythmias and sudden cardiac death. Our previous study of the human ether-à-go-go related gene (hERG)–encoded K+ channel (Kv11.1) supports an association between hERG and RING finger protein 207 (RNF207) variants in aggravating the onset and severity of LQTS, specifically T613M hERG (hERGT613M) and RNF207 frameshift (RNF207G603fs) mutations. However, the underlying mechanistic underpinning remains unknown.

Objective

The purpose of the present study was to test the role of RNF207 in the function of hERG-encoded K+ channel subunits.

Methods

Whole-cell patch-clamp experiments were performed in human embryonic kidney (HEK 293) cells and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) together with immunofluorescent confocal and high resolution microscopy, auto-ubiquitinylation assays, and co-immunoprecipitation experiments to test the functional interactions between hERG and RNF207.

Results

Here, we demonstrated that RNF207 serves as an E3 ubiquitin ligase and targets misfolded hERGT613M proteins for degradation. RNF207G603fs exhibits decreased activity and hinders the normal degradation pathway; this increases the levels of hERGT613M subunits and their dominant-negative effect on the wild-type subunits, ultimately resulting in decreased current density. Similar findings are shown for hERGA614V, a known dominant-negative mutant subunit. Finally, the presence of RNF207G603fs with hERGT613M results in significantly prolonged action potential durations and reduced hERG current in human-induced pluripotent stem cell–derived cardiomyocytes.

Conclusion

Our study establishes RNF207 as an interacting protein serving as a ubiquitin ligase for hERG-encoded K+ channel subunits. Normal function of RNF207 is critical for the quality control of hERG subunits and consequently cardiac repolarization. Moreover, our study provides evidence for protein quality control as a new paradigm in life-threatening cardiac arrhythmias in patients with LQTS.



中文翻译:

破坏人类 ether-à-go-go 相关基因 K+ 通道的蛋白质质量控​​制导致严重的长 QT 综合征

背景

长 QT 综合征 (LQTS) 是一种遗传性疾病,可使患者易患危及生命的心律失常和心源性猝死。我们之前对人类ether-à-go-go 相关基因 ( hERG ) 编码的 K +通道 (K v 11.1) 的研究支持 hERG 和 RING 指蛋白 207 (RNF207) 变体之间的关联在加重 LQTS 的发病和严重性方面,特别是 T613M hERG (hERG T613M ) 和 RNF207 移码 (RNF207 G603fs ) 突变。然而,潜在的机制基础仍然未知。

客观的

本研究的目的是测试 RNF207 在hERG编码的K +通道亚基的功能中的作用。

方法

在人胚胎肾 (HEK 293) 细胞和人诱导多能干细胞衍生的心肌细胞 (hiPSC-CM) 中进行全细胞膜片钳实验,并结合免疫荧光共聚焦和高分辨率显微镜、自泛素化测定和免疫共沉淀测试hERG和RNF207之间功能相互作用的实验。

结果

在这里,我们证明了 RNF207 作为 E3 泛素连接酶并靶向错误折叠的 hERG T613M蛋白进行降解。RNF207 G603fs活性降低,阻碍正常降解途径;这增加了 hERG T613M亚基的水平及其对野生型亚基的显性负效应,最终导致电流密度降低。hERG A614V显示了类似的发现,这是一种已知的显性失活突变亚基。最后,在人诱导的多能干细胞衍生的心肌细胞中,RNF207 G603fs与 hERG T613M的存在导致动作电位持续时间显着延长并降低了 hERG 电流。

结论

我们的研究将 RNF207 确立为一种相互作用的蛋白质,用作hERG编码的K +通道亚基的泛素连接酶。RNF207 的正常功能对于 hERG 亚基的质量控制和因此的心脏复极至关重要。此外,我们的研究为蛋白质质量控​​制作为 LQTS 患者危及生命的心律失常的新范例提供了证据。

更新日期:2021-10-09
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