Division of Cardiovascular Medicine, Department of Internal Medicine, University of California, Davis, Davis, California.
Division of Cardiovascular Medicine, Department of Internal Medicine, University of California, Davis, Davis, California; Department of Physiology and Cell Biology, University of Nevada, Reno, Reno, Nevada.
Heart Rhythm. 2022 Feb;19(2):281-292. doi: 10.1016/j.hrthm.2021.10.005. Epub 2021 Oct 9.
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 (K11.1) supports an association between hERG and RING finger protein 207 (RNF207) variants in aggravating the onset and severity of LQTS, specifically T613M hERG (hERG) and RNF207 frameshift (RNF207) mutations. However, the underlying mechanistic underpinning remains unknown.
The purpose of the present study was to test the role of RNF207 in the function of hERG-encoded K channel subunits.
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.
Here, we demonstrated that RNF207 serves as an E3 ubiquitin ligase and targets misfolded hERG proteins for degradation. RNF207 exhibits decreased activity and hinders the normal degradation pathway; this increases the levels of hERG subunits and their dominant-negative effect on the wild-type subunits, ultimately resulting in decreased current density. Similar findings are shown for hERG, a known dominant-negative mutant subunit. Finally, the presence of RNF207 with hERG results in significantly prolonged action potential durations and reduced hERG current in human-induced pluripotent stem cell-derived cardiomyocytes.
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.
长 QT 综合征（LQTS）是一种遗传性疾病，使患者容易发生危及生命的心律失常和心源性猝死。我们之前对人 ether-à-go-go 相关基因（hERG）编码的 K 通道（K11.1）的研究支持 hERG 与 RING 指蛋白 207（RNF207）变体之间的关联，这些变体加剧了 LQTS 的发病和严重程度，特别是 T613M hERG（hERG）和 RNF207 移码（RNF207）突变。然而，其潜在的机制尚不清楚。
本研究旨在测试 RNF207 在 hERG 编码的 K 通道亚基功能中的作用。
在人胚肾（HEK 293）细胞和人诱导多能干细胞衍生的心肌细胞（hiPSC-CMs）中进行全细胞膜片钳实验，结合免疫荧光共聚焦和高分辨率显微镜、自动泛素化测定和共免疫沉淀实验，以测试 hERG 和 RNF207 之间的功能相互作用。
在这里，我们证明 RNF207 作为一种 E3 泛素连接酶，可将错误折叠的 hERG 蛋白靶向降解。RNF207 活性降低并阻碍正常的降解途径；这会增加 hERG 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。对已知的显性负突变亚基 hERG 也有类似的发现。最后，在人诱导多能干细胞衍生的心肌细胞中，RNF207 与 hERG 的存在导致动作电位持续时间显著延长，hERG 电流减少。
本研究确立了 RNF207 作为 hERG 编码的 K 通道亚基的相互作用蛋白，作为一种泛素连接酶。RNF207 的正常功能对于 hERG 亚基的质量控制和心脏复极至关重要。此外，我们的研究为蛋白质量控制作为 LQTS 患者危及生命的心律失常的新范式提供了证据。
