Structural / functional basis of CaM dependent modulation of NaV1.5 inactivation

描述（由申请人提供）：电压门控钠（NaV）通道是可兴奋细胞中的基本信号分子，决定Na+离子的快速流入。对人类心脏NaV1.5通道（SCN5A基因）功能的干扰可导致心律失常，在某些情况下可导致死亡。SCN5A基因的遗传缺陷是长QT期3型综合征的已知原因，并与Brugada综合征和婴儿猝死综合征等其他危及生命的疾病有关。对于正常功能的NaV通道，细胞内钙（[Ca2+]i）的变化导致NaV1.5的快速失活。已知这一过程涉及细胞内钙敏感蛋白钙调蛋白（CaM）、位于C端附近的通道的特定元件以及结构域3和4之间的连接区域（D3D4连接体）。最近的研究证实D3D4连接子具有快速失活门的功能（图）。在一些情况下，上述突变发生在或可能改变CaM和FIG的相互作用和功能，这是本研究的重点。CaM-FIG相互作用表征的最新进展已经确定了特定残基的突变，这些突变可以从[Ca2+]i依赖性中解耦快速失活门控。然而，Ca2+传感装置在FIG上的作用的分子基础目前是有争议的，很大程度上是未知的。基于相互矛盾的观点，文献中提出了几个模型，但没有产生能够协调所有可用数据的功能模型。我假设[Ca2+] I的变化导致CaM和NaV1.5 c端特定元件的重塑，这导致CaM以先前模型未描述的方式与FIG （D3D4连接器）相互作用。了解CaM-FIG相互作用的分子细节对于建立准确的模型至关重要，该模型描述了Ca2+和cam依赖的NaV1.5快速失活调制。该项目的完成将详细描述涉及CaM和D3D4连接体的高度复杂机制，将[Ca2+]i的变化转化为人类心脏钠通道快速失活门控的调制。本研究将利用生物物理、结构和电生理技术的补充来表征CaM和FIG之间的相互作用，这对这种调节机制至关重要。这些结果将用于确定CaM-FIG复合物突变对NaV1.5快速失活的相对扰动程度。这一信息将有助于了解特定心脏危及生命的心律失常的分子基础，并为评估小分子靶向NaV1.5 [Ca2+]i传感装置治疗某些心律失常综合征的潜在应用奠定广泛的基础。

DESCRIPTION (provided by applicant): Voltage-gated sodium (NaV) channels are fundamental signaling molecules in excitable cells that determine the rapid influx of Na+ ions. Perturbation to the function of human cardiac NaV1.5 channels (SCN5A gene) can result in arrhythmias and in some cases death. Genetic defects in the SCN5A gene are a known cause of long QT type 3 syndromes, and are associated with other life threatening conditions such as Brugada syndrome and sudden infant death syndrome. For normal functioning NaV channels, changes in intracellular calcium ([Ca2+]i) lead to fast inactivation of NaV1.5. This process is known to involve the intracellular calcium sensing protein calmodulin (CaM), specific elements of the channel located near the C terminus, and the linker region between domains 3 and 4 (D3D4 linker). Recent studies have confirmed the D3D4 linker functions as the fast inactivation gate (FIG). In several instances, the above-mentioned mutations occur in, or are likely to alter interactions and functioning of CaM and the FIG, which is the focus of this work. Recent advances in the characterization of CaM-FIG interactions have identified mutations to specific residues that uncouple fast inactivation gating from [Ca2+]i dependence. However, the molecular basis for the action of the Ca2+ sensing apparatus on the FIG is currently debated and largely unknown. Several models have been proposed in the literature based on conflicting views, but no functional model has been generated that is able to reconcile all the available data. I hypothesize that changes in [Ca2+]i lead to remodeling of CaM and specific elements in the C-terminus of NaV1.5, which result in CaM interacting with the FIG (D3D4 linker) in a manner that is NOT described by previous models. Understanding the molecular details of CaM-FIG interactions are essential to the development of an accurate model that describes the Ca2+ and CaM-dependent modulation of NaV1.5 fast inactivation. Completion of this project will provide a detailed description of the highly complex mechanism that involves CaM and the D3D4 linker translating changes in [Ca2+]i into modulation of fast inactivation gating of the human cardiac sodium channel. This investigation will utilize a complement of biophysical, structural, and electrophysiological techniques to characterize interactions between CaM and the FIG that are essential to this regulatory mechanism. The results will be used to determine the relative extent of perturbation to NaV1.5 fast inactivation caused by mutations to the CaM-FIG complex. This information will aid in understanding the molecular basis of specific cardiac life threatening arrhythmias as well as lay a broad foundation for evaluating the potential use of small molecules to target the NaV1.5 [Ca2+]i sensing apparatus for the treatment of certain cardiac arrhythmia syndromes.