Regulation of cardiac ion channel function via allosteric modulators

描述（由申请人提供）：兰尼碱受体（RyR）由4种RyR原聚体和蛋白质组成，与通道的胞质结构域结合，形成大分子信号传导复合物。这一提议解决了变构调节剂调节RyR功能的机制。将检查两种特定形式的变构调节：1）通过1，4-苯并硫氮杂卓衍生物调节通道，其通过变构效应有效影响通道门控; 2）通过与稳定亚基FKBP 12/12.6（钙稳定蛋白1/2）的蛋白质-蛋白质相互作用调节通道。提出了四个目标：目标1：通过增强钙蛋白2与RyR 2结合的小分子对RyR 2的变构调节。将使用重构成平面脂质双层的RyR 2通道来检查1，4-苯并硫氮杂卓衍生物对RyR 2通道功能的影响。将使用光亲和放射性标记鉴别RyR 2上1，4-苯并硫氮杂卓衍生物的结合位点。假设1，4-苯并硫氮杂卓的衍生物结合并变构调节RyR 2和RyR 1的功能。目标二：RyR 2的变构调节作为预防心律失常的机制RyR 2在心房颤动（AF）中是PKA过度磷酸化和“渗漏”的，JTV 519在WT和钙蛋白2 +/-小鼠中预防运动诱导的心律失常，但在钙蛋白2-/-小鼠中不预防，表明这种新型抗心律失常药物的作用机制需要钙蛋白2。该假说是增强钙蛋白2与RyR 2结合的小分子可以通过RyR 2的变构调节来预防心律失常。使用携带与人类心脏性猝死相关的RyR 2突变的遗传小鼠模型，以及模拟组成性PKA磷酸化或非磷酸化RyR 2的RyR 2突变，以及AF和心肌梗死的动物模型，我们将确定增强钙稳定蛋白2与RyR 2的结合是否可以预防心律失常。目的3：稳定钙蛋白2与RyR 2的结合作为治疗心力衰竭（HF）的机制。RyR 2在HF中是PKA过度磷酸化和钙蛋白2耗尽的，JTV 519改善WT和钙蛋白2 +/-小鼠的心脏功能，但在钙蛋白2-/-小鼠中没有。使用遗传小鼠模型和心肌梗死模型，我们将确定使用JTV 519增强钙稳定蛋白2与RyR 2的结合是否通过变构效应修饰RyR 2改善HF中的心脏功能。在HF中，骨骼肌疲劳增加，RyR 1是PKA过度磷酸化和钙稳定蛋白1耗尽，JTV 519可能通过对通道的变构效应诱导钙稳定蛋白1与RyR 1的重新结合。我们将使用心肌梗塞和心力衰竭的动物模型，研究JTV 519是否改善心力衰竭时的骨骼肌功能。这些研究意义重大，因为它们可能导致一种基于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。

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.