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，是膜兴奋性和离子转运疾病治疗的重要靶点。离子通道在细胞膜上形成离子选择孔，使离子在细胞内和细胞外隔室之间有调节的扩散。特定离子通道类型的功能缺陷会引起广泛的疾病，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作为抑制剂-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打开剂方面存在未开发的潜力。在我们提出的研究中，我们将研究最近引入的一种钾通道打开剂（瑞加滨）的详细作用机制，这种药物正在成为许多癫痫患者的有效治疗药物。我们还将研究我们实验室最近研究的第二个实验系统，在这个实验系统中，药物与离子通道相互作用的细微变化可以导致经典离子通道阻滞剂作为通道打开剂。我们预计，通过研究这两种不同的离子通道增强机制，我们将获得详细的药物：离子通道相互作用，使一些化合物作为通道打开剂的重要见解。这些进展将通过确定特定的化学相互作用对其理想效果至关重要，从而为改进现有药物（如瑞加滨）和开发新的离子通道打开剂提供信息。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方法。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细微的改变。这种方法依赖于工程定制的氨基酸，这些氨基酸通常不被我们的遗传物质编码，并设计一种方法将它们整合到活细胞的离子通道中。这被称为非自然氨基酸诱变，我们是少数几个能够成功应用这种方法的实验室之一。主要的优点是它允许对离子通道结构进行详细的“逐个原子”的改变。这极大地扩展了可用于研究离子通道功能的工具，并能够高度特异性地解剖控制离子通道与治疗调节剂（如瑞加滨）相互作用的化学力。除了我们特定的研究目标外，我们希望我们实验室中这些方法的持续发展将对其他对离子通道或其他蛋白质类的结构功能关系感兴趣的小组的研究做出重大贡献。影响：总之，我们的发现资助计划将应用独特的非自然氨基酸诱变技术来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充当离子通道打开剂的详细机制。我们预计这些发现将加速离子通道打开剂的开发和改进，如瑞加滨，以及其他可能有效治疗某些心血管和神经疾病的化合物。

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.