Tuning ion channel:drug interactions with unnatural amino acid mutagenesis

概述：离子通道蛋白是所有细胞电行为的重要调节因子，也是膜兴奋性和离子转运疾病治疗药物开发的重要靶点。离子通道在细胞膜中形成离子选择性孔，使得能够调节细胞内和细胞外区室之间的离子扩散。特定类型离子通道的功能缺陷可导致多种疾病，从心律失常到激素分泌障碍（糖尿病、高胰岛素血症）、耳聋、共济失调等。背景：大多数靶向离子通道蛋白的药物都起抑制剂的作用--它们降低离子通道的整体活性。然而，在开发增加通道活性的化合物或通道开放剂方面存在未开发的潜力。在我们提出的研究中，我们将研究最近推出的钾通道开放剂（瑞替加滨）的详细作用机制，该药物正在成为许多患者癫痫的有效治疗方法。我们还将研究我们实验室的研究最近描述的第二个实验系统，其中药物：离子通道相互作用的微妙变化可以导致经典的离子通道阻滞剂作为通道开放剂。我们预计，通过研究这两种不同的离子通道增强机制，我们将获得重要的见解详细的药物：离子通道相互作用，使一些化合物作为通道开放剂。这些进展将通过确定对其理想效果至关重要的特定化学相互作用，为现有药物（如瑞替加宾）的改进和新离子通道开放剂的开发提供信息。除癫痫外，钾通道开放剂有望成为某些心律失常、高血压和其他疾病的有效治疗药物。为了解决这些问题，我们实施了一种非常独特的实验方法，使我们能够对离子通道结构进行非常微妙的改变。这种方法依赖于工程定制的氨基酸，这些氨基酸通常不是由我们的遗传物质编码的，并设计一种方法将它们纳入活细胞的离子通道。这被称为非天然氨基酸诱变，我们是能够成功应用这种方法的少数实验室之一。主要优点是它允许详细的“原子对原子”的离子通道结构的改变。这极大地扩展了可用于研究离子通道功能的工具的调色板，并且使得能够高度特异性地解剖控制离子通道与治疗调节剂如瑞替加滨相互作用的化学力。除了我们的具体研究目标，我们希望在我们的实验室正在进行的这些方法的发展将显着有助于研究感兴趣的离子通道或其他蛋白质类的结构功能关系的其他团体。影响：总之，我们的发现资助计划将应用独特的非天然氨基酸诱变技术，以确定离子通道和调节剂之间的详细化学相互作用。这项研究计划的长期影响将是确定使某些化合物能够作为离子通道开放剂的详细机制。我们预计，这些见解将加速离子通道开放剂（如瑞替加宾）和其他可能有效治疗某些心血管和神经系统疾病的化合物的开发和改进。

Overview:Ion channel proteins are essential regulators of electrical behavior in all cells, and important targets for the development of therapeutics for diseases of membrane excitability and ion transport. Ion channels form ion-selective pores in cell membranes, enabling regulated diffusion of ions between the intracellular and extracellular compartments. Defects in function of specific ion channel types cause a wide range of disorders, ranging from cardiac arrhythmias, to hormone secretory disorders (diabetes, hyperinsulinism), deafness, ataxias, and others.Background:Most drugs that target ion channel proteins act as inhibitors – they reduce the overall activity of the ion channels. However, there is untapped potential in the development of compounds that increase channel activity, or channel openers. In our proposed research we will investigate the detailed mechanism of action of a recently introduced potassium channel opener (retigabine) that is emerging as an effective treatment for epilepsy in many patients. We will also investigate a second experimental system recently described by research in our lab, in which subtle alterations of drug:ion channel interactions can cause classical ion channel blockers to act as channel openers. We anticipate that by studying these two distinct mechanisms of ion channel potentiation, we will gain important insights into the detailed drug:ion channel interactions that enable some compounds to act as channel openers. These advances will inform the improvement of existing drugs like retigabine, and the development of new ion channel openers, by identifying specific chemical interactions that are essential for their desirable effects. In addition to epilepsy, potassium channel openers are expected to be effective treatments for certain cardiac arrhythmias, hypertension, and other disorders.To approach these problems, we have implemented a very unique experimental method that allows us to make very subtle changes to ion channel structure. This method relies on engineering custom made amino acids that are not normally encoded by our genetic material, and devising a way to incorporate them into ion channels in living cells. This is referred to as unnatural amino acid mutagenesis, and we are one of only a few laboratories that has been able to successfully apply this method. The major advantage is that it allows detailed ‘atom-by-atom’ alteration of ion channel structure. This dramatically expands the palette of tools available to investigate ion channel function, and enables highly specific dissection of chemical forces that control ion channel interactions with therapeutic modulators like retigabine. In addition to our specific research objectives we are hopeful that ongoing development of these methods in our lab will significantly contribute to research by other groups interested in structure function relationships of ion channels or other protein classes. Impact:In summary, our Discovery Grant program will apply unique technology for unnatural amino acid mutagenesis to identify detailed chemical interactions between ion channels and modulators. The long term impact of this research program will be the identification of detailed mechanisms that enable certain compounds to act as ion channel openers. We anticipate that these insights will accelerate the development and improvement of ion channel openers like retigabine, and other compounds that may be effective therapeutics for certain cardiovascular and neurological disorders.