GPCR-Based Regulation of the hERG Potassium Channel Biosynthesis and Function

基于 GPCR 的 hERG 钾通道生物合成和功能调节

• 批准号: 8074427
• 负责人: Yamini A. Krishnan
• 金额: $ 4.68万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8074427
• 关键词: Acute; Address; Adrenergic Agents; Adrenergic Agonists; Adrenergic Receptor; Affect; Anabolism; Arrhythmia; Biochemical; Biosensor; Cardiac; Cardiac Output; Cardiovascular system; Cells; Chronic; Complex; Congestive; Cultured Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dependence; Disease; Dominant-Negative Mutation; Epinephrine; Ethers; Event; Failure; Financial compensation; Fluorescence Resonance Energy Transfer; Future; G Protein-Coupled Receptor Genes; G-Protein-Coupled Receptors; Gases; Genes; Goals; Health; Heart Diseases; Heart Rate; Hormonal; Hormones; Hour; Human; Hypertension; In Vitro; Incidence; Inherited; Ion Channel; Ion Channel Gating; Ischemia; Label; Lead; Link; Long QT Syndrome; Mediating; Mediator of activation protein; Methods; Molecular; Mutation; Myocardium; Norepinephrine; Pathologic; Pathway interactions; Patients; Pharmaceutical Preparations; Phorbol Esters; Phospholipase C; Phosphorylation; Phosphotransferases; Physiological; Potassium; Potassium Channel; Prevention; Process; Protein Isoforms; Proteins; Radioisotopes; Receptor Mediated Signal Transduction; Regulation; Regulatory Pathway; Research; Risk; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; System; Tachyarrhythmias; Testing; Time; Translations; Up-Regulation; Work; Xenopus; adrenergic; base; design; heart rhythm; in vitro Assay; inhibitor/antagonist; mimetics; mutant; protein kinase A kinase; receptor; research study; response; voltage

DESCRIPTION (provided by applicant): Mutations in the hERG (human ether-a-go-go related gene) potassium channel are linked to the hereditary Long QT syndrome (LOTS, locus LQT2). Patients with the hereditary LOTS or drug-induced LOTS are susceptible to fatal tachyarrhythmias. HERG channels are regulated by several intracellular signaling pathways that together contribute to the overall modulation of the potassium current IKr in normal and disease states. Previous studies have established the acute regulation of HERG current and gating though the p-adrenergic system via direct phosphorylation of the HERG channel. Recent studies show that acute treatment of HERG-expressing Xenopus oocyteswith phorbol esters, a broad PKC activator, leads to a shift in the voltage dependence of activation of HERG. Chronic effects of adrenergic stimulation on the HERG channel have not been studied. Our experiments show that 24-hour stimulation with increased intracellular cAMP levels ((3-adrenergicpathway) or phorbol esters (a-adrenergic pathway) result in distinct increases in HERG protein abundance which are not transcriptionally mediated. The goal of the proposed research is to elucidate the mechanisms by which the P- and a-adrenergic pathways (via PKA and PKC) modulate changes in HERG protein abundance. We hypothesize that chronic p-and a-adrenergic receptor stimulation specifically enhances the rate of HERG synthesis through mechanisms involving PKA and the PKC isoforms which ultimately lead to translational up-regulation. We propose to determine the roles of PKA and PKC during translation of HERG using polysomal profiling with quantitative real-time PCR and in-vitro translation methods with radioisotope labeling. We also propose to dissect the upstream adrenergic signaling pathways leading to increases in HERG abundance by using pharmacological and biochemical methods in a heterologous expression system. PUBLIC HEALTH RELEVANCE: Long-term increases in the circulating levels of the hormones epinephrine and norepinephrine are consistently observed in patients with chronic heart disease and this may put them at an increased risk for abnormal heart rhythms. Our research is designed to examine the molecular pathways between the long-term hormonal stimulation and changes in the cardiac HERG ion-channel, which normally helps to maintain the heart rhythm. This work may identify potential targets for therapy in the prevention of rhythm disturbances accompanying chronic heart disease.

描述（由申请方提供）：hERG（人ether-a-go-go相关基因）钾通道突变与遗传性长QT综合征（LOTS，LQT 2位点）相关。遗传性LOTS或药物诱导的LOTS患者易发生致命性快速性心律失常。HERG通道受几种细胞内信号传导途径调节，这些信号传导途径共同促进正常和疾病状态下钾电流IKr的总体调节。先前的研究已经通过HERG通道的直接磷酸化建立了HERG电流和通过β-肾上腺素能系统的门控的急性调节。最近的研究表明，急性治疗HERG表达爪蟾卵母细胞与佛波酯，广泛的PKC激活剂，导致转移的电压依赖性激活HERG。尚未研究肾上腺素能刺激对HERG通道的慢性影响。我们的实验表明，增加细胞内cAMP水平（β-肾上腺素能途径）或佛波酯（α-肾上腺素能途径）的24小时刺激导致HERG蛋白丰度的明显增加，这不是转录介导的。本研究的目的是阐明β-和α-肾上腺素能通路（通过PKA和PKC）调节HERG蛋白丰度变化的机制。我们推测，慢性β-和α-肾上腺素能受体刺激通过涉及PKA和PKC亚型的机制特异性增强HERG合成速率，最终导致翻译上调。我们建议确定PKA和PKC在翻译HERG过程中的作用，使用实时定量PCR和体外翻译方法与放射性同位素标记的多聚体分析。我们还建议通过在异源表达系统中使用药理学和生物化学方法来剖析导致HERG丰度增加的上游肾上腺素能信号传导途径。公共卫生相关性：在慢性心脏病患者中，经常观察到肾上腺素和去甲肾上腺素循环水平的长期增加，这可能会增加他们心律异常的风险。我们的研究旨在研究长期激素刺激与心脏HERG离子通道变化之间的分子途径，这通常有助于维持心律。这项工作可能会确定潜在的治疗目标，在预防心律失常伴随慢性心脏病。