Regulation of cardiac ion channel function via allosteric modulators

通过变构调节剂调节心脏离子通道功能

• 批准号: 7027234
• 负责人: ANDREW Robert MARKS
• 金额: $ 40.25万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-12-20 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7027234
• 关键词: FK506; allosteric site; arrhythmia; binding proteins; calcium channel; calcium flux; cardiovascular disorder prevention; chemoprevention; disease /disorder model; genetically modified animals; heart failure; heart function; intracellular transport; laboratory mouse; muscle contraction; myocardium; phosphorylation; protein kinase A; protein structure function; receptor binding; small molecule; thiazide

DESCRIPTION (provided by applicant): The ryanodine receptor (RyR) is comprised of 4 RyR protomers and proteins that bind to the cytoplasmic domain of the channel forming a macromolecular signaling complex. This proposal addresses the mechanisms by which allosteric modulators regulate RyR function. Two specific forms of allosteric modulation will be examined: 1) regulation of the channel by derivatives of 1,4-benzothiazepines that potently effect channel gating via allosteric effects; 2) regulation of the channel by protein-protein interactions with the stabilizing subunit FKBP12/12.6 (calstabin1/2). Four aims are proposed: Aim 1: Allosteric regulation of RyR2 by small molecules that enhance binding of calstabin2 to RyR2. Effects of 1,4- benzothiazepine derivatives on RyR2 channel function will be examined using RyR2 channels reconstituted into planar lipid bilayers. The binding site on RyR2 for the 1,4-benzothiazepine derivatives will be identified using photoaffinity radiolabels. The hypothesis is derivatives of 1,4-benzothiazepines bind to and allosterically modulate the function of RyR2 and RyR1. Aim 2: Allosteric modulation of RyR2 as a mechanism for preventing cardiac arrhythmias RyR2 are PKA hyperphosphorylated and "leaky" in atrial fibrillation (AF) and JTV519 prevents exercised induced cardiac arrhythmias in WT and calstabin2+/- mice but not in calstabin2-/- mice indicating that the mechanism of action of this novel anti-arrhythmic drug requires calstabin2. The hypothesis is small molecules that enhance calstabin2 binding to RyR2 can prevent cardiac arrhythmias via allosteric modulation of RyR2. Using genetic mouse models harboring RyR2 mutations linked to sudden cardiac death in humans, and RyR2 mutations that mimic constitutively PKA phosphorylated or non-phosphorylatable RyR2 and animal models of AF, and myocardial infarction, we will determine whether enhancing binding of calstabin2 to RyR2 prevents cardiac arrhythmias. Aim 3: Stabilization of calstabin2 binding to RyR2 as a mechanism for treating heart failure (HF). RyR2 are PKA hyperphosphorylated and depleted of calstabin2 in HF and JTV519 improves cardiac function in WT and calstabin2+/- mice but not in calstabin2-/- mice. Using genetic mouse models and models of myocardial infarction, we will determine whether enhancing binding of calstabin2 to RyR2 using JTV519 that modify RyR2 via allosteric effects improve cardiac function in HF. Skeletal muscle fatigue is increased and RyR1 are PKA hyperphosphorylated and depleted of calstabin1 in HF, and JTV519 induces rebinding of calstabin1 to RyR1 probably via an allosteric effect on the channel. Using animal models of myocardial infarction and HF, we will investigate whether JTV519 improves skeletal muscle function in HF. The studies are significant because they may lead to a novel therapeutic approach based on allosteric modulation of RyR that can result in improved therapy for human cardiovascular diseases.

描述（由申请人提供）：兰尼碱受体（RyR）由 4 个 RyR 原聚体和蛋白质组成，它们与通道的细胞质结构域结合，形成大分子信号复合物。该提案阐述了变构调节剂调节 RyR 功能的机制。将检查变构调节的两种具体形式：1）1,4-苯并硫氮杂卓衍生物对通道的调节，通过变构效应有效影响通道门控； 2) 通过与稳定亚基 FKBP12/12.6 (calstabin1/2) 的蛋白质-蛋白质相互作用来调节通道。提出了四个目标： 目标 1：通过增强 calstabin2 与 RyR2 结合的小分子对 RyR2 进行变构调节。将使用重构为平面脂质双层的 RyR2 通道来检查 1,4- 苯并硫氮杂卓衍生物对 RyR2 通道功能的影响。 1,4-苯并硫氮杂卓衍生物在 RyR2 上的结合位点将使用光亲和放射性标记进行鉴定。该假设是 1,4-苯并硫氮杂卓类药物的衍生物与 RyR2 和 RyR1 结合并变构调节其功能。目标 2：RyR2 的变构调节作为预防心律失常的机制 RyR2 是 PKA 过度磷酸化且在心房颤动 (AF) 中“渗漏”，JTV519 可预防 WT 和 calstabin2+/- 小鼠中运动诱发的心律失常，但不能预防 calstabin2-/- 小鼠表明这种新型抗心律失常药物的作用机制需要calstabin2。假设增强 calstabin2 与 RyR2 结合的小分子可以通过 RyR2 的变构调节来预防心律失常。使用携带与人类心源性猝死相关的 RyR2 突变的基因小鼠模型，以及模拟 PKA 磷酸化或非磷酸化 RyR2 的 RyR2 突变的基因小鼠模型以及 AF 和心肌梗塞的动物模型，我们将确定增强 calstabin2 与 RyR2 的结合是否可以预防心脏骤停心律失常。目标 3：稳定 calstabin2 与 RyR2 的结合作为治疗心力衰竭 (HF) 的机制。 RyR2 在 HF 中被 PKA 过度磷酸化并耗尽了 calstabin2，而 JTV519 可改善 WT 和 calstabin2+/- 小鼠的心脏功能，但不会改善 calstabin2-/- 小鼠的心脏功能。使用遗传小鼠模型和心肌梗塞模型，我们将确定使用通过变构效应修饰 RyR2 的 JTV519 增强 calstabin2 与 RyR2 的结合是否可以改善心衰患者的心脏功能。 HF 中骨骼肌疲劳增加，RyR1 被 PKA 过度磷酸化并耗尽 calstabin1，JTV519 可能通过通道的变构效应诱导 calstabin1 与 RyR1 重新结合。我们将使用心肌梗塞和心力衰竭的动物模型来研究 JTV519 是否可以改善心力衰竭时的骨骼肌功能。这些研究意义重大，因为它们可能会带来一种基于 RyR 变构调节的新型治疗方法，从而改善人类心血管疾病的治疗。