Graduate School of Pharmaceutical Sciences, Keio University, Shibakoen, Minato-ku, Tokyo, 105-8512, Japan.
Division of Biophysics and Neurobiology, Department of Molecular and Cellular Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki-shi, Aichi, 444-8585, Japan.
BMC Mol Cell Biol. 2021 Jan 7;22(1):3. doi: 10.1186/s12860-020-00337-3.
Human ether-à-go-go-related gene potassium channel 1 (hERG) is a voltage-gated potassium channel, the voltage-sensing domain (VSD) of which is targeted by a gating-modifier toxin, APETx1. APETx1 is a 42-residue peptide toxin of sea anemone Anthopleura elegantissima and inhibits hERG by stabilizing the resting state. A previous study that conducted cysteine-scanning analysis of hERG identified two residues in the S3-S4 region of the VSD that play important roles in hERG inhibition by APETx1. However, mutational analysis of APETx1 could not be conducted as only natural resources have been available until now. Therefore, it remains unclear where and how APETx1 interacts with the VSD in the resting state.
We established a method for preparing recombinant APETx1 and determined the NMR structure of the recombinant APETx1, which is structurally equivalent to the natural product. Electrophysiological analyses using wild type and mutants of APETx1 and hERG revealed that their hydrophobic residues, F15, Y32, F33, and L34, in APETx1, and F508 and I521 in hERG, in addition to a previously reported acidic hERG residue, E518, play key roles in the inhibition of hERG by APETx1. Our hypothetical docking models of the APETx1-VSD complex satisfied the results of mutational analysis.
The present study identified the key residues of APETx1 and hERG that are involved in hERG inhibition by APETx1. These results would help advance understanding of the inhibitory mechanism of APETx1, which could provide a structural basis for designing novel ligands targeting the VSDs of K channels.
人 Ether-à-go-go 相关基因钾通道 1（hERG）是一种电压门控钾通道，其电压感应域（VSD）是门控调节剂毒素 APETx1 的靶标。APETx1 是海葵 Anthopleura elegantissima 的 42 个残基肽毒素，通过稳定静息状态来抑制 hERG。先前对 hERG 进行半胱氨酸扫描分析的研究确定了 VSD 的 S3-S4 区域中的两个残基，它们在 APETx1 抑制 hERG 中起重要作用。然而，由于直到现在只有天然资源可用，因此无法进行 APETx1 的突变分析。因此，APETx1 在静息状态下与 VSD 相互作用的位置和方式仍不清楚。
我们建立了制备重组 APETx1 的方法，并确定了重组 APETx1 的 NMR 结构，该结构与天然产物结构相当。使用野生型和突变型的 APETx1 和 hERG 进行电生理分析表明，APETx1 中的疏水残基 F15、Y32、F33 和 L34 以及 hERG 中的 F508 和 I521，除了先前报道的酸性 hERG 残基 E518，在 APETx1 抑制 hERG 中起关键作用。我们假设的 APETx1-VSD 复合物对接模型满足突变分析的结果。
本研究确定了 APETx1 和 hERG 中参与 APETx1 抑制 hERG 的关键残基。这些结果将有助于深入了解 APETx1 的抑制机制，为设计针对 K 通道 VSD 的新型配体提供结构基础。
