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: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=587998
• 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，并调节心脏、神经和内分泌组织的兴奋性。在心脏组织中，Kv通道的打开抑制了兴奋性并终止了动作电位。人类以太-a-go-go相关基因（hERG） Kv通道是介导心脏复极的关键因素，hERG功能障碍与心律失常的关联突出了这一点。hERG通道结构与其他Kv通道相似：S1-S6跨膜域，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运动与孔的耦合涉及一个短而重要的细胞质S4- s5连接体。hERG通道还具有n端per - art - sim （PAS）结构域和c端环核苷酸结合结构域（cNBD）。