Tuning ion channel:drug interactions with unnatural amino acid mutagenesis

概述： 离子通道蛋白是所有细胞中电行为的重要调节因子，也是开发膜兴奋性和离子转运疾病治疗药物的重要靶点。离子通道在细胞膜上形成离子选择孔，使离子能够在细胞内和细胞外的隔间进行有控制的扩散。特定离子通道类型的功能缺陷会导致广泛的疾病，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： 大多数针对离子通道蛋白的药物都是抑制剂--它们会降低离子通道的整体活性。然而，在提高通道活性的化合物或通道开放剂的开发方面仍有未开发的潜力。在我们提出的研究中，我们将研究一种最近引入的钾通道开放剂(瑞加宾)的详细作用机制，该药正在成为许多患者的有效治疗癫痫的药物。我们还将研究我们实验室最近描述的第二个实验系统，在该系统中，药物：离子通道相互作用的细微变化可以导致经典的离子通道阻滞剂充当通道开放剂。我们预计，通过研究这两种不同的离子通道增强机制，我们将对详细的药物：使某些化合物能够作为通道开放剂的离子通道相互作用获得重要的见解。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(如雷替加宾)的改进和新离子通道开放剂的开发提供信息。除了癫痫，钾通道开放剂有望成为治疗某些心律失常、高血压和其他疾病的有效药物。 为了解决这些问题，我们实施了一种非常独特的实验方法，允许我们对离子通道结构进行非常细微的改变。这种方法依赖于设计定制的氨基酸，这些氨基酸通常不是由我们的遗传物质编码的，并设计出一种方法，将它们整合到活细胞的离子通道中。这被称为非天然氨基酸突变，我们是少数几个能够成功应用这种方法的实验室之一。它的主要优点是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的调色板，并使控制离子通道与治疗调节剂(如瑞替加宾)相互作用的化学力能够进行高度特异性的剖析。除了我们的具体研究目标外，我们希望我们实验室正在进行的这些方法的开发将对其他对离子通道或其他蛋白质类别的结构功能关系感兴趣的小组的研究做出重大贡献。 影响： 总而言之，我们的发现资助计划将应用独特的技术进行非天然氨基酸突变，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的开发和改进，如雷替加宾和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.