Department of Physiology, University of Maryland School of Medicine, Baltimore, MD.
Department of Physiology, University of Maryland School of Medicine, Baltimore, MD
J Gen Physiol. 2019 Apr 1;151(4):478-488. doi: 10.1085/jgp.201812129. Epub 2018 Nov 13.
Human ether-à-go-go-related gene (hERG, KCNH2) voltage-activated potassium channels are critical for cardiac excitability. hERG channels have characteristic slow closing (deactivation), which is auto-regulated by a direct interaction between the N-terminal Per-Arnt-Sim (PAS) domain and the C-terminal cyclic nucleotide binding homology domain (CNBHD). hERG channels are not activated by the binding of extrinsic cyclic nucleotide ligands, but rather bind an "intrinsic ligand" that is composed of residues 860-862 within the CNBHD and mimics a cyclic nucleotide. The intrinsic ligand is located at the PAS-CNBHD interface, but its mechanism of action in hERG is not well understood. Here we use whole-cell patch-clamp electrophysiology and FRET spectroscopy to examine how the intrinsic ligand regulates gating. To carry out this work, we coexpress PAS (a PAS domain fused to cyan fluorescent protein) in trans with hERG "core" channels (channels with a deletion of the PAS domain fused to citrine fluorescent protein). The PAS domain in trans with hERG core channels has slow (regulated) deactivation, like that of WT hERG channels, as well as robust FRET, which indicates there is a direct functional and structural interaction of the PAS domain with the channel core. In contrast, PAS in trans with hERG F860A core channels has intermediate deactivation and intermediate FRET, indicating perturbation of the PAS domain interaction with the CNBHD. Furthermore, PAS in trans with hERG L862A core channels, or PAS in trans with hERG F860G,L862G core channels, has fast (nonregulated) deactivation and no measurable FRET, indicating abolition of the PAS and CNBHD interaction. These results indicate that the intrinsic ligand is necessary for the functional and structural interaction between the PAS domain and the CNBHD, which regulates the characteristic slow deactivation gating in hERG channels.
人类 ether-à-go-go 相关基因 (hERG, KCNH2) 电压激活钾通道对于心脏兴奋性至关重要。hERG 通道具有特征性的缓慢关闭（失活），这是通过 N 端 Per-Arnt-Sim (PAS) 结构域和 C 端环核苷酸结合同源结构域 (CNBHD) 之间的直接相互作用自动调节的。hERG 通道不被外源性环核苷酸配体的结合激活，而是结合一种“内源性配体”，该配体由 CNBHD 内的残基 860-862 组成，并模拟环核苷酸。内源性配体位于 PAS-CNBHD 界面，但它在 hERG 中的作用机制尚不清楚。在这里，我们使用全细胞膜片钳电生理学和 FRET 光谱学来研究内源性配体如何调节门控。为了进行这项工作，我们在反式共表达 PAS（与青色荧光蛋白融合的 PAS 结构域）与 hERG“核心”通道（与黄色荧光蛋白融合的 PAS 结构域缺失的通道）。反式 PAS 与 hERG 核心通道具有缓慢（调节）失活，与 WT hERG 通道相似，并且具有强大的 FRET，这表明 PAS 结构域与通道核心之间存在直接的功能和结构相互作用。相比之下，反式 PAS 与 hERG F860A 核心通道具有中间失活和中间 FRET，表明 PAS 结构域与 CNBHD 的相互作用受到干扰。此外，反式 PAS 与 hERG L862A 核心通道或反式 PAS 与 hERG F860G,L862G 核心通道具有快速（非调节）失活和不可测量的 FRET，表明 PAS 和 CNBHD 相互作用被废除。这些结果表明，内源性配体是 PAS 结构域和 CNBHD 之间功能和结构相互作用所必需的，这调节了 hERG 通道特征性的缓慢失活门控。
