Mechanisms of regulation of hERG ion channels by cytoplasmic factors

细胞质因子调控hERG离子通道的机制

• 批准号: RGPIN-2014-04759
• 负责人: Claydon, Thomas
• 金额: $ 2.55万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=629602
• 关键词: Mechanisms; regulation; hERG; ion; channels

Ion channels control membrane excitability and are critical determinants of cellular function. Voltage-gated K (Kv) channels, the largest sub-family, are responsive to the transmembrane potential and regulate excitability in cardiac, nerve, and endocrine tissues. In cardiac tissues, opening of Kv channels dampens excitability and terminates the action potential. The human ether-a-go-go related gene (hERG) Kv channel is a critical player mediating cardiac repolarization and this is highlighted by the association of hERG dysfunction with cardiac arrhythmia. hERG channel structure is similar to that of other Kv channels: S1-S6 transmembrane domains with S1-S4 forming the voltage sensor and S5-S6 the pore. S4 contains basic amino acids that sense transmembrane voltage and trigger movement of S4, which opens the pore. Coupling of S4 movement to the pore involves a short, but important, cytoplasmic S4-S5 linker. hERG channels also possess an N-terminal Per-Arnt-Sim (PAS) domain and C-terminal cyclic nucleotide binding domain (cNBD).In contrast to other Kv channels, the gating properties of hERG channels are unique and poorly understood. This affords them their critical role in termination of the action potential. hERG channels activate and deactivate slowly, yet inactivate and recover from inactivation rapidly. Because inactivation is faster than activation, hERG currents during depolarization (early in the action potential) are small, and robust current is only observed upon repolarization (action potential termination) when channels rapidly recover from inactivation, but have yet to close. The slow closing is thus a crucial feature that allows current to flow during repolarization and this is highlighted by the association of mutations that accelerate deactivation gating with cardiac arrhythmia. Despite this, the mechanistic basis of these unusual gating events is poorly understood. Perturbations in hERG gating occur in response to mutations in different regions of the channel: N- and C-terminus, voltage sensing unit and S4-S5 linker. However, the mechanism by which these signals from diverse regions are integrated is unclear. We propose that the cytoplasmic S4-S5 linker plays a key role as an integrator of signals from cytoplasmic channel domains, i.e. N- and C-terminal domains would modify gating by direct interaction with the S4-S5 linker that modulates S4 movement or its coupling to the pore gate. We propose that the S4-S5 linker may integrate numerous cytoplasmic signals in this way.We propose a combined approach of electrophysiology, hypothesis-driven mutagenesis and biochemistry to investigate how hERG function is controlled by cytoplasmic domains of the channel. In Objective 1, we propose to define interactions between the S4-S5 linker and N- and/or C- terminus that regulate hERG channel function. We hypothesize that the S4-S5 linker makes specific key contacts that mediate interaction with one/both of these domains and we will use site-directed mutagenesis and two-electrode voltage clamp to test this. In Objective 2, we propose to measure these interactions directly using isothermal titration calorimetry to detect bi-molecular protein-protein interactions. This approach allows for biophysical measurements of the reaction to inform on the nature of the interactions. Lastly, in Objective 3, we propose to determine the physiological role of the N-terminal PAS domain in hERG channels. Its physiological role in hERG channels is not known, and we intend to investigate a putative role in oxygen and/or redox potential sensing and its transduction to hERG channel functional changes. Thus, our combined technical approach aims to describe the molecular mechanisms by which hERG channels are gated by cytoplasmic signals.

离子通道控制膜的兴奋性，是细胞功能的关键决定因素。电压门控性钾通道（Voltage-gated K（Kv）channels）是最大的钾通道亚家族，对跨膜电位有反应，并调节心脏、神经和内分泌组织的兴奋性。在心脏组织中，Kv通道的开放抑制兴奋性并终止动作电位。人类ether-a-go-go相关基因（hERG）Kv通道是介导心脏复极化的关键参与者，这一点通过hERG功能障碍与心律失常的关联而突出。hERG通道结构类似于其他Kv通道：S1-S6跨膜结构域，其中S1-S4形成电压传感器，S5-S6形成孔。S4含有碱性氨基酸，可以感知跨膜电压并触发S4的运动，从而打开孔。S4运动与孔的偶联涉及短但重要的细胞质S4-S5接头。hERG通道还具有N-末端Per-Arnt-Sim（PAS）结构域和C-末端环核苷酸结合结构域（cNBD）。这使它们在终止动作电位中发挥关键作用。hERG通道激活和失活缓慢，但失活和恢复迅速。由于失活快于激活，去极化（动作电位早期）期间的hERG电流较小，仅在通道从失活中快速恢复但尚未关闭时，在复极化（动作电位终止）时观察到稳健电流。因此，缓慢闭合是允许电流在复极期间流动的关键特征，并且这通过加速失活门控与心律失常的突变的关联而突出。尽管如此，这些不寻常的门控事件的机制基础是知之甚少。hERG门控中的扰动响应于通道的不同区域中的突变而发生：N-和C-末端、电压感应单元和S4-S5接头。然而，这些来自不同区域的信号被整合的机制尚不清楚。我们提出，胞质S4-S5连接子作为来自胞质通道结构域的信号的整合者起着关键作用，即N-和C-末端结构域将通过与调节S4运动或其与孔门的偶联的S4-S5连接子直接相互作用来修改门控。我们提出S4-S5连接子可能以这种方式整合了许多胞质信号，我们提出了一种电生理学、假设驱动的诱变和生物化学相结合的方法来研究hERG功能是如何由该通道的胞质结构域控制的。在目标1中，我们提出定义S4-S5接头与调节hERG通道功能的N-和/或C-末端之间的相互作用。我们假设S4-S5连接子产生介导与这些结构域中的一个/两个相互作用的特定关键接触，并且我们将使用定点诱变和双电极电压钳来测试这一点。在目标2中，我们建议直接使用等温滴定量热法来测量这些相互作用，以检测双分子蛋白质-蛋白质相互作用。这种方法允许对反应进行生物物理测量，以了解相互作用的性质。最后，在目标3中，我们提出确定N-末端PAS结构域在hERG通道中的生理作用。其在hERG通道中的生理作用尚不清楚，我们打算研究氧和/或氧化还原电位传感及其转导到hERG通道功能变化中的假定作用。因此，我们的组合技术方法旨在描述hERG通道通过细胞质信号门控的分子机制。