Modulators of hERG/HCN4 Function and Block

HERG是一种K通道，调节自律性、心脏发生、血管发生和神经发生。它调节癌症的转移。我的研究项目描述了受内源性细胞环境因素调节的HERG的结构决定因素，如phi、亲脂物质、异构体和常见的多态。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基于结晶化hEAG的HERG新原子学模型：2016年8月，神经元hEAG通道的高分辨率冷冻-EM结构已被解决。我们最初的硅基HERG模型是基于Kv1.2晶体。HEAG和HERG非常同源，但Kv1.2明显不同。我们正在基于hEAG创建一个新的HERG模型。*Aim 2a亲脂通路：我们发现M651T突变使伊夫拉定阻断HERG电流的IC_(50)从6µM/L移至120µM/L，但不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部位的亲脂性途径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。这些药剂能阻断海尔格电流。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。*Aim 2b细胞内pH：在《自然》的科学报告中，我们展示了细胞内pH的变化对多非利特反应的HERG功能的影响。我们建议评估Phi对神经酰胺、鞘氨醇-1-P和胆固醇等内源性物质阻断HERG的影响。神经酰胺在生理pH下是可电离的，而胆固醇则不是。目的3 HERG不同亚型常见多态对内源性物质反应的影响：编码HERG的KCNH2基因存在常见多态。常见的多态性包括K897T、R1047L和Y652a。WT和含有多态的通道的反应将被检测为对亲脂性内源性物质的反应。以前的研究表明，K897T突变改变了QT对缺血的反应，形成了研究Phi的基础。达夫实验室首先发现了Herg1b亚型。这些多态在不同的亚型中的影响还没有被探索。此外，我们看到Y652a在不同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模型跨膜结构域的胞内末端。我们的电子计算机研究表明，在S2-S3和S4-S5连接子中存在PAS结构域的受体。*我们的程序提供了关于为什么这种K通道的不同亚型或共同的多态变体对共同的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.