Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel.
J Gen Physiol. 2020 Apr 6;152(4). doi: 10.1085/jgp.201912505.
Human KCNH2 channels (hKCNH2, ether-à-go-go [EAG]-related gene, hERG) are best known for their contribution to cardiac action potential repolarization and have key roles in various pathologies. Like other KCNH family members, hKCNH2 channels contain a unique intracellular complex, consisting of an N-terminal eag domain and a C-terminal cyclic nucleotide-binding homology domain (CNBHD), which is crucial for channel function. Previous studies demonstrated that the CNBHD is occupied by an intrinsic ligand motif, in a self-liganded conformation, providing a structural mechanism for the lack of KCNH channel regulation by cyclic nucleotides. While there have been significant advancements in the structural and functional characterization of the CNBHD of KCNH channels, a high-resolution structure of the hKCNH2 intracellular complex has been missing. Here, we report the 1.5 Å resolution structure of the hKCNH2 channel CNBHD. The structure reveals the canonical fold shared by other KCNH family members, where the spatial organization of the intrinsic ligand is preserved within the β-roll region. Moreover, measurements of small-angle x-ray scattering profile in solution, as well as comparison with a recent NMR analysis of hKCNH2, revealed high agreement with the crystallographic structure, indicating an overall low flexibility in solution. Importantly, we identified a novel salt-bridge (E807-R863) which was not previously resolved in the NMR and cryo-EM structures. Electrophysiological analysis of charge-reversal mutations revealed the bridge's crucial role in hKCNH2 function. Moreover, comparison with other KCNH members revealed the structural conservation of this salt-bridge, consistent with its functional significance. Together with the available structure of the mouse KCNH1 intracellular complex and previous electrophysiological and spectroscopic studies of KCNH family members, we propose that this salt-bridge serves as a strategically positioned linchpin to support both the spatial organization of the intrinsic ligand and the maintenance of the intracellular complex interface.
人类 KCNH2 通道（hKCNH2，与醚-α--go-go [EAG]相关基因，hERG）以其对心脏动作电位复极化的贡献而闻名，并在各种病理中发挥关键作用。与其他 KCNH 家族成员一样，hKCNH2 通道包含一个独特的细胞内复合物，由一个 N 端 eag 结构域和一个 C 端环核苷酸结合同源结构域（CNBHD）组成，这对通道功能至关重要。以前的研究表明，CNBHD 被一个内在的配体基序占据，处于自配体构象，为缺乏环核苷酸对 KCNH 通道调节提供了结构机制。虽然 KCNH 通道的 CNBHD 的结构和功能特征已经有了显著的进展，但 hKCNH2 细胞内复合物的高分辨率结构仍然缺失。在这里，我们报告了 hKCNH2 通道 CNBHD 的 1.5 Å 分辨率结构。该结构揭示了其他 KCNH 家族成员所共有的典型折叠，其中内在配体的空间组织在β-滚区域内得以保留。此外，溶液中小角 X 射线散射谱的测量以及与最近 hKCNH2 的 NMR 分析的比较，与晶体结构高度一致，表明在溶液中整体灵活性较低。重要的是，我们发现了一个新的盐桥（E807-R863），这在以前的 NMR 和 cryo-EM 结构中没有得到解决。对电荷反转突变的电生理分析表明，该桥在 hKCNH2 功能中起着至关重要的作用。此外，与其他 KCNH 成员的比较表明，该盐桥的结构保守，与其功能意义一致。结合现有的小鼠 KCNH1 细胞内复合物结构以及以前对 KCNH 家族成员的电生理和光谱研究，我们提出，该盐桥作为一个战略性的连接点，支撑内在配体的空间组织和细胞内复合物界面的维持。
