Role of Segment 6 in Heart Na Channel Slow Inactivation

6 段在心脏 Na 通道缓慢失活中的作用

• 批准号: 6899539
• 负责人: JOHN P O'REILLY
• 金额: $ 17.91万
• 依托单位: SOUTHEASTERN LOUISIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6899539
• 关键词: cardiovascular agents; conformation; electrophysiology; gene mutation; heart function; membrane activity; membrane structure; protein structure function; site directed mutagenesis; sodium channel; sudden cardiac death; transfection; voltage gated channel

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to better understand the relationship between molecular structure and physiologic function in voltage-gated sodium channels (Navs). The specific aims focus on the role of two regions of cardiac Navs (hNavl.5), transmembrane segment 6 in domains 1 and 2 (D1-S6 and D2-S6), in an electrophysiologic property called slow inactivation. Navs are transmembrane proteins that play a critical role in the normal electrophysiology of excitable tissues, and proper function of Navs is dependent on specific structural characteristics. Amino acid mutations in Navs can alter normal physiologic function, and Nav mutations have been identified that underlie such human conditions as epilepsy, muscle myotonias, and sudden cardiac death syndrome. Several of these mutations affect slow inactivation and thereby alter membrane excitability and normal electrophysiologic function. This proposal addresses the hypothesis that conformational changes in D1-S6 and/or D2-S6 play an important role in the process of slow inactivation. To address this hypothesis, site-directed mutagenesis (alanine, lysine, or cysteine substitutions), a cellular expression system (HEK cells), and patch-clamp techniques will be used to study slow inactivation of wildtype and mutant hNav1.5. The SCAM (substituted-cysteine accessibility method) technique using methanethiosulfonate (MTS) agents and the cysteine-substituted mutants will be employed to look for molecular movement in these regions associated with slow inactivation. Experiments will be performed to compare slow inactivation in wild-type and mutants Navs. Cysteine-substituted mutants will be studied before and after MTS exposure using specific voltage protocols to assess state-dependent (e.g., slowinactivated) effects on MTS accessibility. The results will provide valuable information on molecular mechanisms and protein conformational changes during slow inactivation of Navs. This information will be useful for understanding heart Nav channelopathies such as long QT and Brugada syndromes.

描述(由申请人提供)：该提案的长期目标是更好地了解电压门控钠通道(NAV)中分子结构和生理功能之间的关系。具体目标集中在心脏NAV的两个区域(hNav1.5)，结构域1和2的跨膜段6(D1-S6和D2-S6)在一种称为缓慢失活的电生理特性中所起的作用。NAV是一种跨膜蛋白，在可兴奋组织的正常电生理中起着至关重要的作用，其正常功能依赖于特定的结构特征。NAV中的氨基酸突变可以改变正常的生理功能，NAV突变已被发现是癫痫、肌强直和心源性猝死综合征等人类疾病的基础。这些突变中有几个会影响缓慢的失活，从而改变膜的兴奋性和正常的电生理功能。这一建议解决了D1-S6和/或D2-S6的构象变化在缓慢失活过程中发挥重要作用的假说。为了解决这一假设，将使用定点突变(丙氨酸、赖氨酸或半胱氨酸替代)、细胞表达系统(HEK细胞)和膜片钳技术来研究野生型和突变型hNav1.5的缓慢失活。使用甲硫磺酸盐(MTS)试剂和半胱氨酸取代突变体的SCAM(取代半胱氨酸可及性方法)技术将被用来寻找这些与缓慢失活相关的区域中的分子运动。将进行实验来比较野生型和突变型Navs的缓慢失活。半胱氨酸替代突变体将在MTS暴露前后使用特定的电压方案进行研究，以评估状态依赖(例如，缓慢失活)对MTS可及性的影响。这些结果将为研究NAV在缓慢失活过程中的分子机制和蛋白质构象变化提供有价值的信息。这些信息将有助于了解心脏导航通道病变，如长QT和Brugada综合征。