Role of Segment 6 in Heart Na Channel Slow Inactivation

项目描述（申请人提供）：该项目的长期目标是对电压门控Na+通道（Navs）的分子结构与生理功能之间的关系有一个基本的了解。具体而言，本项目将重点研究人类心脏Nav异构体hNav1.5。了解hNav1.5正常结构和功能之间的关系将有助于深入了解异常结构和功能之间的关系，例如在由nav遗传突变（通道病变）引起的心脏病中发现的异常结构和功能之间的关系。该项目的具体目标将集中在hNav1.5的缓慢失活，这是一个动力学过程，在动作电位放电特性和设置膜兴奋性中很重要。位点定向诱变、HEK细胞中表达的野生型和突变型hNav1.5的电生理记录，以及取代半胱氨酸接近法（SCAM）将用于确定内孔区域（D1-S6和D2-S6）在hNav1.5缓慢失活中的功能作用。具体目标1将确定大小和疏水性不同的半胱氨酸(C)和谷氨酰胺(Q)取代D1-S6和D2-S6内孔区域的天然氨基酸对hNav1.5中缓慢失活的影响。这些替换将跨越D1-S6中的N406和D2-S6中的V930到各自结构域S6中假定的门控铰链的区域。全细胞Na+电流的电生理记录将用于确定取代的功能效果。假设内孔的这个区域在缓慢失活门控中是关键的，因此，该区域的突变将破坏正常的缓慢失活。特异性Aim 2将确定在hNav1.5缓慢失活期间D1-S6和/或D2-S6的内孔区域是否存在分子重排。假设该区域的构象变化是缓慢失活的重要分子机制，并且这种分子重排改变了这些区域中关键氨基酸的相对位置。这一假设将使用来自Specific Aim 1的半胱氨酸取代突变体通道，用取代半胱氨酸接近性方法（SCAM）进行验证。半胱氨酸取代的突变体将在静止、快速失活和缓慢失活状态下暴露于甲乙硫磺酸（MTS）试剂中。mts可达性将被用作D1-S6和D2-S6在缓慢失活期间相对位置变化和移动的指标。这一建议将为Nav缓慢失活的分子机制提供新的信息，这将增强我们对长QT期和Brugada综合征等以Nav缓慢失活等动力学过程破坏为特征的人类心脏疾病的理解。公共卫生相关性：在长QT期综合征和Brugada综合征患者中发现了80多种心脏钠通道突变。这些突变可引起心脏正常电生理功能的改变，从而导致成人和婴儿的心源性猝死（即婴儿猝死综合征，SIDS）。了解心脏钠通道分子结构与生理功能之间的关系，将有助于我们了解人类心脏的异常功能（病理生理学），这可能为心脏性猝死的诊断、治疗干预和/或预防提供有用的信息。

DESCRIPTION (provided by applicant): The long-term objective of this project is to acquire a fundamental understanding of the relationship between molecular structure and physiological function in voltage-gated Na+ channels (Navs). Specifically, this project will focus on the human cardiac Nav isoform hNav1.5. Understanding the relationship between normal structure and function in hNav1.5 will provide insight into the relationship between abnormal structure and function such as that found in cardiac diseases resulting from heritable mutations in Navs (channelopathies). The specific aims of this project will focus on slow inactivation in hNav1.5, a kinetic process that is important in action potential firing properties and setting membrane excitability. Site-directed mutagenesis, electrophysiological recordings from wild-type and mutant hNav1.5 expressed in HEK cells, and the substituted-cysteine accessibility method (SCAM) will be used to determine the functional role of the inner pore region (of D1-S6 and D2-S6) in hNav1.5 slow inactivation. The following specific aims will be addressed: Specific Aim 1 will determine the effect on slow inactivation in hNav1.5 of substituting cysteine (C) and glutamine (Q), which vary in size and hydrophobicity, for the native amino acids in the inner pore region of D1-S6 and D2-S6. The substitutions will span the region from N406 in D1-S6 and V930 in D2-S6 to the putative gating-hinges in S6 of the respective domains. Electrophysiological recordings of whole-cell Na+ current will be used to determine the functional effect of the substitutions. The hypothesis is that this region of the inner pore is critical in slow inactivation gating and therefore, that mutagenesis in this region will disrupt normal slow inactivation. Specific Aim 2 will determine if there is molecular rearrangement in the inner pore regions in D1-S6 and/or in D2-S6 during slow inactivation in hNav1.5. The hypothesis is that conformational changes in this region are an important molecular mechanism of slow inactivation and that this molecular rearrangement alters the relative positions of critical amino acids in these regions. This hypothesis will be tested with the substituted-cysteine accessibility method (SCAM) using the cysteine-substituted mutant channels from Specific Aim 1. The cysteine-substituted mutants will be exposed to methanethiosulfonate (MTS) reagents at rest, during fast inactivation, and while in the slow-inactivated state. MTS-accessibility will be used as an indicator of relative positional changes and movement in D1-S6 and D2-S6 during slow inactivation. This proposal will provide novel information on the molecular mechanism of Nav slow inactivation, which will enhance our understanding of human heart diseases such as long QT and Brugada Syndrome that are characterized by disruption of Nav kinetic processes such as slow inactivation. PUBLIC HEALTH RELEVENCE: More than 80 mutations in human heart sodium channels have been identified in patients with conditions such as long QT and Brugada syndrome. These mutations can produce changes in the normal electrophysiological function of the heart, which can lead to sudden cardiac death in adults and infants (i.e., sudden infant death syndrome, SIDS). Understanding the relationship between molecular structure and physiological function in cardiac sodium channels will provide us with an understanding of abnormal function (pathophysiology) of the human heart, which may provide useful information for diagnosis, therapeutic intervention, and/or prevention of sudden cardiac death.