Using a Molecular Toolkit to Examine Potassium Channel Gating and Regulation

使用分子工具包检查钾通道门控和调节

• 批准号: 10534951
• 负责人: Miranda Schene
• 金额: $ 3.75万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10534951
• 关键词: Address; Adrenergic Agents; Affect; Amino Acids; Anions; Arrhythmia; Binding; Cardiac Myocytes; Cells; Charge; Consensus; Contracts; Core Protein; Cyclic AMP-Dependent Protein Kinases; Data; Dependence; Development; Disabled Persons; Electrostatics; Fluorine; Fluorometry; Functional disorder; Future; Goals; Heart; Heart Rate; Hydrophobicity; Immobilization; Individual; Inherited; Investigation; Ion Channel; Ion Channel Gating; Light; Long QT Syndrome; Mammalian Cell; Measures; Molecular; Movement; Mutagenesis; Mutate; Mutation; Outcome; Pharmacology; Phenylalanine; Phosphorylation; Phosphorylation Site; Physicians; Physiologic pulse; Physiological; Play; Positioning Attribute; Potassium; Potassium Channel; Proteins; Regulation; Research; Resolution; Rest; Role; Scientist; Serine; Signal Transduction; Site; Stimulus; Structure; Sympathetic Nervous System; Voltage-Gated Potassium Channel; Work; career; disorder prevention; drug development; effective therapy; heart function; new therapeutic target; novel; novel therapeutics; patch clamp; photolysis; prevent; response; sensor; sudden cardiac death; training opportunity; unnatural amino acids; voltage; voltage clamp

Project Summary/Abstract The IKS channel is a voltage-gated potassium channel found in the heart. After a cardiac muscle cell is depolarized, the IKS channel opens and allows a potassium current to leave the cell, returning it to resting state. Dysfunction in this channel is associated with numerous acquired and inherited arrhythmias, including long QT syndome, a leading cause of sudden cardiac death. The channel itself is made of two protein components: KCNQ1 (Q1), the pore-forming subunit; and KCNE1 (E1), a single transmembrane accessory protein. When Q1 is expressed alone, the channel opens at a negative voltage and conducts a small potassium current, but with E1 bound, the channel opens at a high positive voltage and conducts a large potassium current. Thus E1 acts as an intrinsic gating regulator of the channel. The IKS channel is also regulated transiently by phosphorylation in response to signaling by the sympathetic nervous system. When phosphorylated, the channel opens more quickly and more often, which leads to an increaed potassium current. This leads to faster cell repolarization and facilitates increased heart rate. However, the molecular mechanisms of both intrinsic regulation by E1 and transient regulation by phosphorylation are not currently understood. The goal of this research is to gain a high- resolution understanding of these mechanisms of regulation. Intrinsically, phenylalanine (Phe) has been shown to play a role in many ion channel mechanisms because despite being overall nonpolar and largely hydrophobic, it has a quadropole moment that creates a negative charge in the center of its aromatic ring. This means that it can behave like an anion and form strong charge-charge interactions with other charged residues, despite being in the hydrophobic core of the channel. Preliminary data has been found that shows a particular Phe residue in the IKS channel participates in a previously uncharacterized charge interaction that slows IKS channel response to voltage, and is thus a key player in intrinsic regulation. This work will characterize this interaction, determine how E1 binding alters it, and determine the specifc role this interaction plays in regulating IKS channel function. As well as intrinsic regulation by E1, Q1 is transiently regulated by phosphorylation at two sites on the N-terminus. Cumulatively, the channel response to phosphorylation is an increase in current, but the mechanism of this increase is unknown. Additionally, the individual roles of the two phosphorylation sites are not understood. This research will use caged serine, a modified serine residue that can only be phosphorylated after photolysis of a bound caging moiety. Encoding caged serine into each of the sites individually and observing the effects of phosphorylation at each site on the channel will provide this key information about phosphoregulation of the IKS channel. This work, which will provide mechanistic explanations of intrinsic and transient regulation of the IKS channel, will pave the way for development of novel therapies for long QT syndrome and other arrythmias attributed to this channel.

项目摘要/摘要 IKS通道是在心脏中发现的电压门控钾通道。心肌细胞是 去极化，IKS通道打开并允许钾电流离开电池，将其返回到静止状态。 该通道中的功能障碍与许多获得和遗传的心律不齐有关，包括长QT Syndome，这是心脏突然死亡的主要原因。该通道本身由两个蛋白质成分组成： KCNQ1（Q1），孔形成亚基；和KCNE1（E1），单个跨膜辅助蛋白。 Q1 单独表示，该通道以负电压打开并导致小钾电流，但有 E1结合，通道以高正电压打开，并导致大钾电流。因此，E1行为 作为通道的内在门控调节剂。 IKS通道还通过磷酸化瞬时调节 响应交感神经系统的信号。当磷酸化时，通道打开更多 迅速，更频繁地，导致钾电流增加。这会导致更快的细胞复极化，并 促进心率增加。但是，E1和 目前尚不清楚通过磷酸化的瞬时调节。这项研究的目的是获得高 解决这些调节机制的理解。本质上，已经显示了苯丙氨酸（PHE） 在许多离子通道机制中发挥作用 它具有四倍时刻，在其芳香环中心产生负电荷。这意味着 尽管有一个阴离子的表现，但与其他充电残留物形成强烈的充电相互作用 在通道的疏水核心中。发现初步数据显示了特定的PHE残基 IKS频道参与先前未表征的电荷交互 电压，因此是内在调节的关键参与者。这项工作将表征这种互动，确定 E1的结合如何改变它，并确定该相互作用在调节IKS通道函数中发挥的指定作用。 除E1的固有调节外，Q1在N末端的两个位点瞬时受到磷酸化的瞬时调节。 累积地，通道对磷酸化的响应是电流的增加，但是这种机制 增加是未知的。另外，尚不清楚两个磷酸化位点的个别作用。这 研究将使用笼丝氨酸，这是一种修饰的丝氨酸残基，只有在光解之后才能磷酸化 绑定的笼子部分。将笼丝氨酸分别编码到每个站点中，并观察 通道上每个位点的磷酸化将提供有关IKS磷化调节的关键信息 渠道。这项工作将提供IKS内在和瞬态调节的机械解释 渠道，将为长QT综合征和其他雅利氏症的新疗法开发铺平道路 归因于此频道。