Structural Determinants of PIP2 Regulation

Kir3或GIRK（G蛋白门控内向整流K+）通道由迷走神经激活以控制心脏 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， 具有快速调整心率所需的适应性。GIRK通道是有吸引力的药物靶点 心房颤动（AF）是最常见的心律失常，其患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。目前的抗结核药物缺乏特异性， 使用会造成心室副作用的显著风险。这使得相当有吸引力的目标表示 主要在心房。心脏GIRK通道的过度活跃与氧化应激有关。 通过蛋白激酶C（PKC）酶的失调，如 增加新PKCe的活性。然而，即使GIRK活性的完全抑制剂可以逆转AF， 也会抑制心率变异性，这是一种对心脏健康有害的副作用。因此，需要特定的部分抑制剂， 逆转PKC介导的通道激活但不抑制通道的重要功能的药物是未满足的 医疗需求。在这个建议中，我们研究了PKC依赖性磷酸化影响 活性，并表明它变构影响通道与PIP2的相互作用，PIP2是通道的主要调节因子。 膜蛋白功能我们确定了一个PKCe用来刺激通道活性的磷酸化位点 并建议确定所有涉及的网站，并确定门，他们变构耦合导致 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，其也通过PIP2变构控制不同的通道门。在这 我们的目标是将小分子活性调节剂的分子见解与 特异性逆转PKC介导的通道活性过度刺激。我们的小分子抑制剂 在PKC介导的AF的转基因模型中进行了测试，目的是降低异常活性，足以纠正 AF问题而不损害心脏健康。

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.