Modulators of hERG/HCN4 Function and Block

hERG/HCN4 功能和模块的调节剂

• 批准号: RGPIN-2017-03766
• 负责人: Duff, Henry
• 金额: $ 2.04万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=682584
• 关键词: Modulators; hERG; HCN4; Function; Block

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.

hERG是调节自律性、心脏发生、血管发生和神经突发生的K通道。它调节癌症转移。 我的研究项目表征了由内源性细胞环境因素（如pHi、亲脂性物质、同种型变体和常见多态性）调节的hERG的结构决定因素。 我们的工作将提供深入了解为什么这种K通道的变体表现出对内源性因素的差异反应。** 目标1基于结晶hEAG的hERG新原子模型：神经元hEAG通道的高分辨率cryo-EM结构已于2016年8月解析。我们最初的hERG二氧化硅模型是基于Kv1.2晶体。hEAG和hERG具有很高的同源性，但Kv1.2明显不同。 我们正在创建一个基于hEAG的hERG新模型。** 目标2a亲脂性通路：我们发现M651 T突变将伊伐拉定诱导的hERG电流阻断的IC 50从6 µM/L变为120 µM/L，但未改变对多非利特的反应。此外，我们实验室的计算机模拟研究表明，M651是亲脂性药物进入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种进入途径对内源性亲脂性物质（包括神经酰胺、鞘氨醇-1-P和胆固醇）的影响。这些药物阻断hERG电流。我们的初步数据表明，特定的M651位点决定神经酰胺的结合。** 目标2b细胞内pH：在Nature's Sci Reports中，我们显示了细胞内pH变化对多非利特响应的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下可电离，而胆固醇则不可。** 目的3 hERG不同亚型的常见多态性对内源性物质反应的影响：编码hERG的KCNH 2基因存在常见多态性。常见的多态性包括K897 T、R1047 L和Y 652 A。将检查WT和含有通道的多态性对亲脂性内源性物质的响应。先前的研究表明，K897 T突变改变了QT对缺血的反应，并形成了研究pHi的基本原理。Duff实验室首先发现了hERG 1b亚型。尚未探索这些多态性在不同亚型中的影响。此外，我们看到Y 652 A在各种亚型中的影响存在令人兴奋的差异。在主题3中，我们还将PAS结构域对接到hERG模型的跨膜结构域的细胞内末端。我们的计算机模拟研究表明，在S2-S3和S4-S5接头中存在PAS结构域的受体。**我们的计划提供了见解，为什么不同的亚型或常见的多态性变异的K通道表现出差异敏感的共同环境因素。