Structural Determinants of PIP2 Regulation

迷走神经激活Kir3或GIRK(G蛋白门控向内整流钾)通道来控制心脏 费率。它们是心率变异性(HRV)的关键决定因素，HRV是心脏健康的指标，赋予心脏 有了适应性，它需要对心率进行快速调整。GIRK通道是有吸引力的药物靶点 抗房颤(AF)，这是最常见的心律失常，其发病率随着年龄的增长而增加 增加死亡、中风和心肌梗死的风险。当前抗心律失常药物缺乏特异性 使用对脑室副作用有很大风险。这使得相当有吸引力的目标被表达出来 主要在心房。氧化应激与心脏GIRK通道过度激活有关 通过蛋白激酶C(PKC)酶的失调而表现出的衰老状态，如 新型PKCE的活性增加。然而，即使完全抑制GIRK活性可以逆转房颤，他们 也会抑制心率变异性，这是一种损害心脏健康的副作用。因此，需要特定的部分抑制剂 认为逆转PKC介导的通道激活但不抑制通道的重要功能是一个未曾满足的问题 医疗需要。在这个方案中，我们研究了PKC依赖的磷酸化影响的机制。 活性并表明它以变构方式影响通道与PIP2的相互作用，PIP2是 膜蛋白功能。我们确定了PKCE用来刺激通道活性的一个磷酸化位点 并提议确定所有涉及的位置，并确定它们与变构耦合导致的门 通道激活。与此同时，我们开发了强大的结构计算模型，使我们能够测试 小分子抑制剂的作用，它也通过PIP2变构控制不同的通道门。在这 提案，我们的目标是将小分子活性调节器的分子洞察力耦合到 具体地说，逆转PKC介导的通道活性过度刺激。我们的小分子抑制剂将是 在PKC介导的房颤转基因模型中进行测试，目的是降低异常活动，以纠正 在不损害心脏健康的情况下解决房颤问题。

Kir3 or GIRK (G protein gated inwardly rectifying K+) channels are activated by the vagus nerve to control heart rate. They are critical determinants of heart rate variability (HRV), an index of cardiac health, endowing the heart with the adaptability it needs to make rapid adjustments in heart rate. GIRK channels are attractive drug targets against atrial fibrillation (AF), the most common arrhythmia whose prevalence increases with age with an increased risk of mortality, stroke and myocardial infarction. The lack of specificity of the current antiarrhythmics used poses significant risk for ventricular side effects. This makes rather attractive targets expressed predominantly in the atria. Overactivity of cardiac GIRK channels has been implicated under the oxidative stress conditions characteristic of aging through a dysregulation of Protein Kinase C (PKC) enzymes, such as the increase in activity of the novel PKCe. Yet, even though full inhibitors of GIRK activity could reverse AF, they would also inhibit HRV, a side effect detrimental to cardiac health. Thus, the need for specific partial inhibitors that reverse the PKC-mediated channel activation but do not inhibit the vital functions of the channel is an unmet medical need. In this proposal, we investigate the mechanism by which PKC-dependent phosphorylation affects activity and show that it allosterically affects the interactions of the channel with PIP2, the master regulator of membrane protein function. We identify one phosphorylation site used by PKCe to stimulate channel activity and propose to determine all the sites involved and identify the gates they allosterically couple with to cause channel activation. In parallel, we have developed powerful structural computational models that allow us to test the action of small molecule inhibitors, which also allosterically control distinct channel gates via PIP2. In this proposal, we aim to set the stage in coupling the molecular insights of small molecule regulators of activity to specifically reverse the PKC-mediated overstimulation of channel activity. Our small molecule inhibitors will be tested in transgenic models of PKC-mediated AF with the goal to dial down the aberrant activity enough to correct the AF problem without compromising cardiac health.