Molecular Pharmacology of An Inherited Heart Disease

描述（由申请人提供）：本申请中提出的研究的总体目标是了解至少部分由SCN5A基因遗传突变引起的心律失常的分子和结构基础，并确定新的靶向突变治疗策略来治疗心律失常。中心假设是这些心律失常发生的一个步骤是由SCN5A基因产物Nav1.5的生物物理性质改变引起的膜电活动的扰动。大多数与疾病相关的改变通道门控的Nav.15突变影响通道失活。本申请中提出的研究的总体目标是了解至少部分由SCN5A遗传突变引起的心律失常的分子和结构基础，SCN5A是编码Nav1.5的基因，Nav1.5是主要的心脏电压门控Na+通道的α亚基，并确定新的靶向突变治疗策略来治疗心律失常。这项研究建立在先前资助期所做的工作的基础上，并继续关注由遗传性Nav1.5突变引起的心律失常的分子基础，但强调Nav1.5羧基末端（C-T）结构域在这些心律失常中的作用。在过去，我们依靠同源模型来深入了解可能导致突变改变通道功能的结构机制。对HEK 293细胞中突变体Nav1.5通道的生理和药理学特性的分析将辅以心肌细胞中的表达，以提供一个框架来测试在生理相关环境中异种表达系统中研究的预测结果。这个项目有两个具体目标。[[第一个目的是通过使用x射线晶体学直接确定在通道失活门缺失和存在的情况下Nav1.5 C-T的三维结构，来测试失活门与C-T结构域形成复合物的假设。]我们将进一步验证Nav1.5 C-T结构域的结构基序是维持该复合体所必需的假设，以及C-T结构的破坏以及与失活门相互作用的确定位点破坏Nav1.5通道失活的假设。[]第二个目的是研究LQT-3和BrS综合征Nav1.5突变的突变特异性药理学和生物物理后果，并确定这些突变通道在HEK 293细胞和心肌细胞中的生理后果和药理学调节。我们假设从这些细胞和分子实验中获得的信息可以直接转化为基于突变基因产物的特定特性的人类治疗干预，并且还揭示了这些和其他由于Na+通道失活缺陷而导致的心脏遗传性疾病的可能相互关系。

DESCRIPTION (provided by applicant): The overall goal of the research proposed in this application is to understand the molecular and structural basis of cardiac arrhythmias caused, at least in part, by inherited mutations of the SCN5A gene, and to determine novel mutation-targeted therapeutic strategies to treat them. The central hypothesis is that one step in the genesis of these arrhythmias is the perturbation of membrane electrical activity caused by alteration in the biophysical properties of the SCN5A gene product, Nav1.5. Most disease-associated Nav.15 mutations that alter channel gating affect channel inactivation. The overall goal of the research proposed in this application is to understand the molecular and structural basis of cardiac arrhythmias caused, at least in part, by inherited mutations of SCN5A, the gene coding for Nav1.5, the alpha subunit of the primary heart Voltage-gated Na+ channel, and to determine novel mutation-targeted therapeutic strategies to treat them. The research proposed builds upon work done during the previous funding period and continues to focus upon the molecular basis of cardiac arrhythmias caused by inherited Nav1.5 mutations, but emphasizes the role of the Nav1.5 carboxy terminal (C-T) domain in these arrhythmias. In the past we have relied on homology models to gain insight into structural mechanisms that might contribute to mutation-altered channel function. Analysis of the physiological and pharmacological properties of mutant Nav1.5 channels in HEK 293 cells will be complemented by expression in cardiac myocytes to provide a framework to test the predictions of studies in heterologous expression systems with consequences in a physiologically relevant environment. There are two specific aims of this project. [[The first aim is to test the hypothesis that the inactivation gate forms a complex with the C-T domain by directly determining the three dimensional structure of the Nav1.5 C-T in the absence and presence of the channel inactivation gate using x-ray crystallography. We will further test the hypothesis that predicted structural motifs of the Nav1.5 C-T domain are necessary to maintain this complex and that disruption of C-T structure as well as identified sites of interaction with the inactivation gate disrupt Nav1.5 channel inactivation.]] The second aim is to investigate mutation-specific pharmacology and biophysical consequences of LQT-3 and BrS Syndrome Nav1.5 mutations and to determine the physiological consequences and pharmacological modulation of these mutant channels in HEK 293 cells and in cardiac myocytes. We hypothesize that information gained from these cellular and molecular experiments can be translated directly to improved therapeutic intervention in humans based on specific properties of mutant gene products, and also shed light on the possible interrelationship of these and other inherited disorders in the heart due to defects in Na+ channel inactivation.