Role of Segment 6 in Heart Na Channel Slow Inactivation

描述(由申请人提供)：本项目的长期目标是对电压门控钠离子通道(NAV)的分子结构和生理功能之间的关系有一个基本的了解。具体地说，这个项目将专注于人类心脏NAV亚型hNav1.5。了解hNav1.5中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，例如由Navs(通道病)的可遗传突变引起的心脏病中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中的缓慢失活，这是一个在动作电位激发特性和设置膜兴奋性方面重要的动力学过程。我们将利用定点突变、野生型和突变型hNav1.5在HEK细胞中表达的电生理记录，以及取代半胱氨酸可及性方法(SCAM)来确定hNav1.5的内孔区(D1-S6和D2-S6)在hNav1.5缓慢失活中的功能作用。将涉及以下特定目标：特定目标1将确定用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内孔区的天然氨基酸对hNav1.5中缓慢失活的影响。替换将跨越从D1-S6中的N406和D2-S6中的V930到各自结构域的S6中可能的门控铰链的区域。全细胞Na+电流的电生理记录将被用来确定取代的功能效应。假设内孔的这一区域在缓慢失活门控中是关键的，因此，该区域的突变将扰乱正常的缓慢失活。特定目标2将确定在hNav1.5的缓慢失活过程中，D1-S6和/或D2-S6的内部孔区是否存在分子重排。假设该区域的构象变化是缓慢失活的重要分子机制，这种分子重排改变了关键氨基酸在这些区域的相对位置。这一假设将用取代半胱氨酸可及性方法(SCAM)进行验证，该方法使用来自特定目标1的半胱氨酸取代突变体通道。半胱氨酸取代突变体将在静止、快速失活和缓慢失活状态下暴露于甲硫磺酸盐(MTS)试剂。MTS-可达性将被用作在缓慢失活期间，在D1-S6和D2-S6中的相对位置变化和移动的指标。这一提议将为NAV缓慢失活的分子机制提供新的信息，这将加深我们对长QT和Brugada综合征等人类心脏疾病的理解，这些疾病的特征是NAV动力学过程的中断，如缓慢失活。公共卫生研究进展：在患有长QT和Brugada综合征等疾病的患者中，已发现人类心脏钠通道有80多个突变。这些突变会改变心脏的正常电生理功能，从而可能导致成人和婴儿的心脏性猝死(即婴儿猝死综合征，SID)。了解心脏钠通道的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)，从而为心脏猝死的诊断、治疗和/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.