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, Tom
• 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: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=566721
• 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K(Kv)通道是最大的亚家族，对跨膜电位有反应，调节心脏、神经和内分泌组织的兴奋性。在心脏组织中，Kv通道的开放抑制了兴奋性并终止了动作电位。人类乙醚-去相关基因(HERG)Kv通道是介导心脏复极的关键角色，这一点在HERG功能障碍与心律失常的关联中得到了强调。HERG通道的结构与其他Kv通道相似：S1-S6跨膜区，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，从而打开毛孔。S4运动与毛孔的偶联涉及一个短小但重要的细胞质S4-S5连接子。HERG通道还具有N端Per-Arnt-Sim(PAS)结构域和C端环核苷酸结合域(CNBD)。与其他Kv通道相比，HERG通道的选通特性是独一无二的，人们对它的了解也很少。这就赋予了它们在终止动作电位中的关键作用。HERG通道激活和失活缓慢，但失活和迅速从失活中恢复。因为失活比激活更快，所以去极化期间(动作电位早期)的HERG电流很小，只有在通道从失活快速恢复但尚未关闭的复极化(动作电位终止)时才能观察到强劲的电流。因此，缓慢关闭是允许电流在复极过程中流动的关键特征，这一点在加速失活门控与心律失常的突变的关联中得到了强调。尽管如此，这些不寻常的门控事件的机制基础却鲜为人知。HERG门控中的扰动响应于通道不同区域的突变：N端和C端、电压传感单元和S4-S5连接子。然而，这些来自不同地区的信号整合的机制尚不清楚。我们认为，细胞质S4-S5连接子作为细胞质通道结构域信号的积分器起着关键作用，即N-末端和C-末端结构域通过与S4-S5连接子的直接相互作用来调节门控，S4-S5连接子调节S4的运动或其与孔门的耦合。我们认为S4-S5连接子可能通过这种方式整合了大量的细胞质信号。我们提出了一种电生理学、假设驱动的突变和生物化学相结合的方法来研究HERG功能是如何由通道的细胞质结构域控制的。在目标1中，我们建议定义S4-S5连接子与调节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连接子进行特定的关键接触，介导与这些结构域中的一个/两个的相互作用，我们将使用定点突变和双电极电压钳来测试这一点。在目标2中，我们建议使用等温滴定量热法直接测量这些相互作用，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，我们建议确定N-末端PAS结构域在HERG通道中的生理作用。它在HERG通道中的生理作用尚不清楚，我们打算研究它在氧和/或氧化还原电位感知中的可能作用以及它对HERG通道功能变化的转导。因此，我们的联合技术方法旨在描述HERG通道被细胞质信号门控的分子机制。