Two mutations at different positions in the CNBH domain of the hERG channel accelerate deactivation and impair the interaction with the EAG domain
Two mutations at different positions in the CNBH domain of the hERG channel accelerate deactivation and impair the interaction with the EAG domain
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DOI：
10.1113/jp276208
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发表时间：
2018-10-01
影响因子：
5.5
通讯作者：
Kubo, Yoshihiro
中科院分区：
文献类型：
--
作者：
Kume, Shinichiro;Shimomura, Takushi;Kubo, Yoshihiro
人ether-a-go-go相关基因（hERG）通道显示出特征性的缓慢失活，并且N末端胞质ether-a-go-go（EAG）结构域和C末端胞质环核苷酸（CN）结合同源性（CNBH）结构域两者的贡献是众所周知的。已知这些域之间的相互作用对于缓慢失活至关重要。我们分析了CNBH结构域及其上游C-接头结构域的突变对缓慢失活的影响，以及EAG和CNBH结构域之间的相互作用，通过电生理和荧光共振能量转移（FRET）分析，使用非洲爪蟾卵母细胞和HEK 293 T细胞表达系统。我们首先观察到CNBH结构域中Phe 860的突变加速失活并消除结构域间的相互作用，据报道CNBH结构域作为内在配体填充CN结合口袋。接下来，我们观察到，在C-接头结构域中Arg 696和Asp 727之间的盐桥没有形成，据报道，该盐桥对于CN调节的通道的功能至关重要。我们新发现了一个对慢失活至关重要的静电相互作用对：Asp 727和Arg 752在CNBH结构域。它们的突变也损害了结构域间的相互作用。综合这些结果，hERG通道中固有配体（Phe 860）和新鉴定的盐桥对（Asp 727和Arg 752）的突变加速了失活，并降低了EAG和CNBH结构域之间的相互作用。未检测到两个结构域之间FRET效率的电压依赖性变化。结果表明，CNBH结构域有助于通过涉及EAG结构域的机制缓慢失活hERG通道。
The human ether-a-go-go related gene (hERG) channel shows characteristic slow deactivation, and the contribution of both of the N-terminal cytoplasmic ether-a-go-go (EAG) domain and the C-terminal cytoplasmic cyclic nucleotide (CN) binding homology (CNBH) domain is well known. The interaction between these domains is known to be critical for slow deactivation. We analysed the effects of mutations in the CNBH domain and its upstream C-linker domain on slow deactivation and the interaction between the EAG and CNBH domains by electrophysiological and fluorescence resonance energy transfer (FRET) analyses using Xenopus oocyte and HEK293T cell expression systems. We first observed that mutations of Phe860 in the CNBH domain, which is reported to fill the CN binding pocket as an intrinsic ligand, accelerate deactivation and eliminate the inter-domain interaction. Next, we observed that the salt bridge between Arg696 and Asp727 in the C-linker domain, which is reported to be critical for the function of CN-regulated channels, is not formed. We newly identified an electrostatically interacting pair critical for slow deactivation: Asp727 and Arg752 in the CNBH domain. Their mutations also impaired the inter-domain interaction. Taking these results together, both mutations of the intrinsic ligand (Phe860) and a newly identified salt bridge pair (Asp727 and Arg752) in the hERG channel accelerated deactivation and also decreased the interaction between the EAG and CNBH domains. Voltage-dependent changes in FRET efficiency between the two domains were not detected. The results suggest that the CNBH domain contributes to slow deactivation of the hERG channel by a mechanism involving the EAG domain.