Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University, 86-1, Nishimachi, Yonago, Tottori 683-8503, Japan.
Department of Physiology II, Kanazawa Medical University, 1-1 Daigaku, Uchinada-machi, Kahoku-gun, Ishikawa 920-0293, Japan.
J Mol Cell Cardiol. 2018 Feb;115:158-169. doi: 10.1016/j.yjmcc.2018.01.009. Epub 2018 Feb 4.
The human ether-a-go-go-related gene (hERG) encodes the α subunit of a rapidly activating delayed-rectifier potassium (I) channel. Mutations of the hERG cause long QT syndrome type 2 (LQT2). Acetylation of lysine residues occurs in a subset of non-histone proteins and this modification is controlled by both histone acetyltransferases and deacetylases (HDACs). The aim of this study was to clarify effects of HDAC(s) on wild-type (WT) and mutant hERG proteins. WThERG and two trafficking-defective mutants (G601S and R752W) were transiently expressed in HEK293 cells, which were treated with a pan-HDAC inhibitor Trichostatin A (TSA) or an isoform-selective HDAC6 inhibitor Tubastatin A (TBA). Both TSA and TBA increased protein levels of WThERG and induced expression of mature forms of the two mutants. Immunoprecipitation showed an interaction between HDAC6 and immature forms of hERG. Coexpression of HDAC6 decreased acetylation and, reciprocally, increased ubiquitination of hERG, resulting in its decreased expression. siRNA against HDAC6, as well as TBA, exerted opposite effects. Immunochemistry revealed that HDAC6 knockdown increased expression of the WThERG and two mutants both in the endoplasmic reticulum and on the cell surface. Electrophysiology showed that HDAC6 knockdown or TBA treatment increased the hERG channel current corresponding to the rapidly activating delayed-rectifier potassium current (I) in HEK293 cells stably expressing the WT or mutants. Three lysine residues (K116, K495 and K757) of hERG were predicted to be acetylated. Substitution of these lysine residues with arginine eliminated HDAC6 effects. In HL-1 mouse cardiomyocytes, TBA enhanced endogenous ERG expression, increased I, and shortened action potential duration. These results indicate that hERG is a substrate of HDAC6. HDAC6 inhibition induced acetylation of hERG which counteracted ubiquitination leading its stabilization. HDAC6 inhibition may be a novel therapeutic option for LQT2.
人类 ether-a-go-go 相关基因（hERG）编码快速激活延迟整流钾（I）通道的α亚基。hERG 的突变导致长 QT 综合征 2 型（LQT2）。赖氨酸残基的乙酰化发生在一组非组蛋白蛋白中，这种修饰受组蛋白乙酰转移酶和去乙酰化酶（HDACs）的控制。本研究旨在阐明 HDAC（s）对野生型（WT）和突变 hERG 蛋白的影响。WThERG 和两个转运缺陷突变体（G601S 和 R752W）在 HEK293 细胞中瞬时表达，并用 pan-HDAC 抑制剂 Trichostatin A（TSA）或同工型选择性 HDAC6 抑制剂 Tubastatin A（TBA）处理。TSA 和 TBA 均增加了 WThERG 的蛋白水平，并诱导了两个突变体的成熟形式的表达。免疫沉淀显示 HDAC6 与 hERG 的不成熟形式相互作用。HDAC6 的共表达降低了 hERG 的乙酰化，反之，增加了 hERG 的泛素化，导致其表达减少。针对 HDAC6 的 siRNA 以及 TBA 则产生相反的效果。免疫化学显示，HDAC6 敲低增加了内质网和细胞表面上 WThERG 和两个突变体的表达。电生理学显示，HDAC6 敲低或 TBA 处理增加了稳定表达 WT 或突变体的 HEK293 细胞中 hERG 通道电流，对应于快速激活延迟整流钾电流（I）。hERG 的三个赖氨酸残基（K116、K495 和 K757）被预测为乙酰化。这些赖氨酸残基被精氨酸取代消除了 HDAC6 的作用。在 HL-1 小鼠心肌细胞中，TBA 增强了内源性 ERG 的表达，增加了 I，并缩短了动作电位持续时间。这些结果表明 hERG 是 HDAC6 的底物。HDAC6 抑制诱导 hERG 的乙酰化，抵消了泛素化导致其稳定。HDAC6 抑制可能是 LQT2 的一种新的治疗选择。
