Regulation of cardiac ion channel function via allosteric modulators

描述（由申请人提供）：ryanodine受体（RyR）由4个RyR原体和蛋白质组成，它们与通道的细胞质结构域结合，形成大分子信号复合物。本研究提出了变构调制剂调节RyR功能的机制。将研究两种特定形式的变构调节：1)通过变构效应有效影响通道门控的1,4-苯并噻唑类衍生物对通道的调节；2)通过与稳定亚基FKBP12/12.6 （calstabin1/2）的蛋白-蛋白相互作用调节通道。目的1：通过小分子增强钙稳定蛋白2与RyR2的结合对RyR2进行变构调节。1,4-苯并噻唑类衍生物对RyR2通道功能的影响将通过将RyR2通道重构为平面脂质双层来研究。1,4-苯并噻唑类衍生物在RyR2上的结合位点将使用光亲和放射性标记进行鉴定。假设是1,4-苯并噻唑类衍生物结合并变构调节RyR2和RyR1的功能。目的2:RyR2的变体调节作为预防心律失常的机制，RyR2在房颤（AF）中PKA过度磷酸化和“泄漏”，JTV519在WT和calstabin2+/-小鼠中预防运动引起的心律失常，但在calstabin2-/-小鼠中没有，这表明这种新型抗心律失常药物的作用机制需要calstabin2。假设是增强钙稳定蛋白2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。利用携带与人类心源性猝死相关的RyR2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RyR2的RyR2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RyR2的结合是否能预防心律失常。目的3：稳定钙稳定蛋白2与RyR2结合作为治疗心力衰竭（HF）的机制。RyR2在HF和JTV519中被PKA过度磷酸化和钙稳定蛋白2的缺失，可以改善WT和钙稳定蛋白2+/-小鼠的心功能，但对钙稳定蛋白2-/-小鼠没有改善作用。通过小鼠遗传模型和心肌梗死模型，我们将确定JTV519通过变构作用修饰RyR2，从而增强calstabin2与RyR2的结合是否能改善心功能。HF患者骨骼肌疲劳增加，RyR1被PKA过度磷酸化，钙稳定蛋白1被耗尽，JTV519可能通过通道的变构效应诱导钙稳定蛋白1与RyR1的重新结合。通过心肌梗死和心衰动物模型，我们将探讨JTV519是否能改善心衰骨骼肌功能。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.