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

描述（由申请人提供）：hERG（人类以太-a-go-go相关基因）钾通道突变与遗传性长QT综合征（LOTS，基因座LQT2）有关。遗传性lot或药物性lot患者易发生致死性心动过速。HERG通道受几种细胞内信号通路的调节，这些信号通路共同促进了正常和疾病状态下钾电流IKr的整体调节。先前的研究已经通过HERG通道的直接磷酸化，建立了HERG电流的急性调节和通过p-肾上腺素能系统的门控。最近的研究表明，用phorbol酯（一种广泛的PKC激活剂）急性治疗表达HERG的爪蟾卵母细胞，可导致HERG激活的电压依赖性转变。肾上腺素能刺激对HERG通道的慢性影响尚未研究。我们的实验表明，24小时刺激增加细胞内cAMP水平（3-肾上腺素能途径）或phorbol酯（a-肾上腺素能途径）导致HERG蛋白丰度明显增加，这不是转录介导的。本研究的目的是阐明P-和a-肾上腺素能通路（通过PKA和PKC）调节HERG蛋白丰度变化的机制。我们假设慢性p-和a-肾上腺素能受体刺激通过涉及PKA和PKC亚型的机制特异性地提高HERG的合成速率，最终导致翻译上调。我们建议使用实时荧光定量PCR的多染色体分析和放射性同位素标记的体外翻译方法来确定PKA和PKC在HERG翻译过程中的作用。我们还建议通过在异源表达系统中使用药理学和生化方法来剖析导致HERG丰度增加的上游肾上腺素能信号通路。公共卫生相关性：慢性心脏病患者体内肾上腺素和去甲肾上腺素的循环水平长期升高，这可能使患者心律异常的风险增加。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，心脏HERG离子通道通常有助于维持心律。这项工作可能会确定预防慢性心脏病伴发心律失常的潜在治疗靶点。

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.