Photo-crosslinking hERG channels causes a U.V.-driven, state-dependent disruption of kinetics and voltage dependence of activation.
Photo-crosslinking hERG channels causes a U.V.-driven, state-dependent disruption of kinetics and voltage dependence of activation.
复制标题
光交联 hERG 通道会导致紫外线驱动的状态依赖性动力学破坏和电压依赖性激活。
DOI：
10.1101/2024.01.09.574834
复制
发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Trudeau,MatthewC
中科院分区：
文献类型：
--
作者：
Codding,SaraJ;Trudeau,MatthewC
人类 ether-à-go-go 相关基因 (hERG) 电压激活钾通道对于心脏兴奋性至关重要。 hERG 的特征性缓慢关闭（失活）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来调节的。我们的目标是了解孔远端的 PAS 结构域如何在门控过程中重新排列，以变构调节通道孔（和离子通量）。为了实现这一目标，我们利用了非经典氨基酸 4-苯甲酰基-L-苯丙氨酸 (BZF)，它是一种光激活交联探针，当受到紫外线 (U.V.) 照射时，它会形成双自由基，能够与含有 C-H 键的基团形成共价交联，从而实现离子通道动力学的选择性和有效的紫外线驱动的光失活。在这里，我们使用 TAG 密码子抑制技术将 BZF 直接整合到 hERG 钾通道 PAS 结构域的三个位置（G47、F48 和 E50）。将 BZF 纳入 PAS 结构域的 hERG 通道 (hERG-BZF) 显示通道的生物物理特性发生显着变化。 hERG-G47BZF 在关闭状态 (-100mV) 下照射时缓慢激活，但在打开 (0mV) 和关闭状态下照射时快速失活。 hERG-F48BZF 通道显示状态独立且 U.V.通道激活（减慢）和通道失活（加速）的剂量依赖性变化，以及电导电压依赖性的显着变化（右移）。当在 -100 mV 下照射时，hERG-E50BZF 显示出状态依赖性和 U.V.通道激活（减慢）和通道失活（加速）的剂量依赖性变化，以及电导电压依赖性的显着变化（右移），仅当通道在关闭状态（−100mV）下受到照射时才发生。这种方法表明，hERG 通道中 PAS 结构域的直接光交联会导致生物物理参数发生可测量的变化，并更广泛地稳定通道的关闭状态。我们认为通道门控的改变是由于光化学交联导致 hERG PAS 域动态运动减少的直接结果。
Human ether-à-go-go related gene (hERG) voltage-activated potassium channels are critical for cardiac excitability. Characteristic slow closing (deactivation) in hERG is regulated by direct interaction between the N-terminal Per-Arnt-Sim (PAS) domain and the C-terminal cyclic nucleotide binding homology domain (CNBHD). We aim to understand how the PAS domain that is distal to the pore rearranges during gating to allosterically regulate the channel pore (and ion flux). To achieve this, we utilized the non-canonical amino acid 4-Benzoyl-L-phenylalanine (BZF) which is a photo-activatable cross-linkable probe, that when irradiated with ultraviolet (U.V.) light forms a double radical capable of forming covalent cross-links with C-H bond-containing groups, enabling selective and potent U.V.-driven photoinactivation of ion channel dynamics. Here we incorporate BZF directly into the hERG potassium channel PAS domain at three locations (G47, F48, and E50) using TAG codon suppression technology. hERG channels with BZF incorporated into the PAS domain (hERG-BZF) showed a significant change in the biophysical properties of the channel. hERG-G47BZF activated slowly when irradiated in the closed state (−100mV) but deactivated quickly when irradiated in both the open (0mV) and closed state. hERG-F48BZF channels showed a state independent and U.V. dose-dependent change in channel activation (slowing down) and channel deactivation (speeding up), as well as a marked change (right-shift) in the voltage-dependence of conductance. When irradiated at −100 mV hERG-E50BZF showed a state dependent and U.V. dose-dependent change in a channel activation (slowing down) and deactivation (speeding up) of channel deactivation, as well as a marked change (right-shift) in the voltage-dependence of conductance that occurred only when the channel was irradiated in the closed state (−100mV). This approach demonstrated that direct photo-crosslinking of the PAS domain in hERG channels causes a measurable change in biophysical parameters and more broadly stabilized the closed state of the channel. We propose that altered channel gating is as a direct result of reduced dynamic motions in the PAS domain of hERG due to photo-chemical crosslinking.