Modulators of hERG/HCN4 Function and Block

hERG是一个调节自动性、心脏发生、血管发生和神经发生的K通道。它调节癌症转移。我的研究项目描述了由内源性细胞环境因素（如pHi、亲脂物质、异构体变体和常见多态性）调节的hERG的结构决定因素。我们的工作将深入了解为什么这种K通道的变体对内源性因素表现出不同的反应。目的1：基于晶体化hEAG的hERG原子模型：2016年8月，研究人员获得了神经元hEAG通道的高分辨率冷冻电镜结构。我们最初的hERG硅内模型是基于Kv1.2晶体的。hEAG和hERG是同源的，但Kv1.2明显不同。我们正在以hEAG为基础，创造一种新的hERG模式。Aim 2a亲脂通路：我们发现M651T突变将伊伐布雷定诱导的hERG电流阻断的IC50从6µM/L转移到120µM/L，但没有改变对多非利特的反应。此外，我们实验室的计算机研究表明，M651是亲脂性药物到达其结合位点的亲脂性通路的关键结构决定因素。我们现在建议评估这一途径对内源性亲脂物质的影响，包括神经酰胺、鞘氨醇-1- p和胆固醇。这些药物阻断hERG电流。我们的初步数据表明，特定的M651位点决定了神经酰胺的结合。目的2b细胞内pH值：在Nature’s Sci Reports中，我们展示了细胞内pH值变化对多非利特对hERG功能的影响。我们建议评估pHi对内源性物质（如神经酰胺、鞘氨醇-1- p和胆固醇）阻断hERG的影响。神经酰胺在生理pH值下可电离，而胆固醇则不能。目的3:hERG不同同工异构体的共同多态性对内源物质反应的影响：编码hERG的KCNH2基因存在共同多态性。常见多态性包括K897T、R1047L；和Y652A。WT的响应和多态性包含通道将检查响应亲脂性内源性物质。先前的研究表明，K897T突变改变了缺血时的QT反应，并形成了研究pHi的基础。Duff实验室首先发现了hERG 1b亚型。这些多态性在不同同种异构体中的影响尚未被探索。此外，我们还发现Y652A在不同同工异构体中的影响存在令人兴奋的差异。在主题3中，我们还将pas结构域与hERG模型跨膜结构域的胞内端对接。我们的计算机研究表明，在S2-S3和S4-S5连接体中存在PAS结构域受体。我们的程序提供了为什么这个K通道的不同同工异构体或共同多态性变异对共同环境因素表现出不同的敏感性的见解。

hERG is a K channel regulating automaticity, cardiogenesis, vasculogenesis and neuritogenesis. It modulates cancer metastasis. My research program characterizes the structural determinants of hERG modulated by endogenous cellular environmental factors such as pHi, lipophilic substances, isoform variants, and common polymorphisms. Our work will provide insight into why variants of this K channel manifest differential response to endogenous factors. Aim 1 New Atomistic Model of hERG based on crystalized hEAG: A high-resolution cryo-EM structure of neuronal hEAG channel has been resolved in Aug 2016. Our original in silica model of hERG was based on the Kv1.2 crystal. The hEAG and hERG are very homologous but Kv1.2 is distinctly different. We are creating a new model of hERG based on hEAG. Aim 2a Lipophilic Access Paths: We discovered that the M651T mutation shifts the IC50 for ivabradine-induced block of the hERG current from 6 µM/L to 120 µM/L, but did not alter response to dofetilide. Moreover, in silico studies from our lab indicate that M651 is a key structural determinant of lipophilic access path for lipophilic drugs to their binding site. We now propose to assess the impact of this access path to endogenous lipophilic substances including ceramide, sphingosine-1-P, and cholesterol. These agents block the hERG current. Our preliminary data indicate that the specific M651 site determines binding of ceramide. Aim 2b Intracellular pH: In Nature's Sci Reports we show the impact of changes in intracellular pH on hERG function in response to dofetilide. We propose to assess the impact of pHi on block of hERG by endogenous substances such as ceramide, sphingosine-1-P, and cholesterol. Ceramide is ionizable at physiologic pH whereas cholesterol is not. Aim 3 Impact of Common Polymorphism in Different Isoforms of hERG on Response to Endogenous Substances: Common polymorphisms exist in the KCNH2 gene, which encodes hERG. Common polymorphisms include K897T, R1047L; and Y652A. Responses of WT and polymorphism containing channels will be examined in response to lipophilic endogenous substances. Previous studies indicate that the K897T mutation alters QT response to ischemia and forms the rationale to study pHi. Duff lab first discovered the hERG 1b isoform. The impact of these polymorphisms in the different isoforms has not been explored. Moreover, we see exciting differences in the impact of Y652A in the various isoforms. In theme 3 we also dock the PAS-domain to the intracellular ends of the transmembrane domains of hERG model. Our in silico studies suggest the presence of a receptor for the PAS domain in the S2-S3 and the S4-S5 linkers.Our program provides insights on why different isoforms or common polymorphic variants of this K channel manifest differential sensitive to common environmental factors.