Abstract：
SCN2A gene-related early-infantile developmental and epileptic encephalopathy (EI-DEE) is a rare and severe disorder that manifests in early infancy. SCN2A mutations affecting the fast inactivation gating mechanism can result in altered voltage dependence and incomplete inactivation of the encoded neuronal Nav1.2 channel and lead to abnormal neuronal excitability. In this study, we evaluated clinical data of seven missense Nav1.2 variants associated with DEE and performed molecular dynamics simulations, patch-clamp electrophysiology, and dynamic clamp real-time neuronal modelling to elucidate the molecular and neuron-scale phenotypic consequences of the mutations. The N1662D mutation almost completely prevented fast inactivation without affecting activation. The comparison of wild-type and N1662D channel structures suggested that the ambifunctional hydrogen bond formation between residues N1662 and Q1494 is essential for fast inactivation. Fast inactivation could also be prevented with engineered Q1494A or Q1494L Nav1.2 channel variants, whereas Q1494E or Q1494 K variants resulted in incomplete inactivation and persistent current. Molecular dynamics simulations revealed a reduced affinity of the hydrophobic IFM-motif to its receptor site with N1662D and Q1494L variants relative to wild-type. These results demonstrate that the interactions between N1662 and Q1494 underpin the stability and the orientation of the inactivation gate and are essential for the development of fast inactivation. Six DEE-associated Nav1.2 variants, with mutations mapped to channel segments known to be implicated in fast inactivation were also evaluated. Remarkably, the L1657P variant also prevented fast inactivation and produced biophysical characteristics that were similar to those of N1662D, whereas the M1501 V, M1501T, F1651C, P1658S, and A1659 V variants resulted in biophysical properties that were consistent with gain-of-function and enhanced action potential firing of hybrid neurons in dynamic action potential clamp experiments. Paradoxically, low densities of N1662D or L1657P currents potentiated action potential firing, whereas increased densities resulted in sustained depolarization. Our results provide novel structural insights into the molecular mechanism of Nav1.2 channel fast inactivation and inform treatment strategies for SCN2A-related EI-DEE. The contribution of non-inactivating Nav1.2 channels to neuronal excitability may constitute a distinct cellular mechanism in the pathogenesis of SCN2A-related DEE.
摘要：
SCN2A基因相关的早期婴儿发育性脑病和癫痫性脑病（EI-DEE）是一种罕见且严重的疾病，表现在婴儿早期。影响快速失活门控机制的SCN2A突变可导致电压依赖性改变和编码的神经元Nav1.2通道的不完全失活，并导致异常的神经元兴奋性。在这项研究中,我们评估了与DEE相关的七个错义Nav1.2变体的临床数据，并进行了分子动力学模拟，膜片钳电生理学，和动态钳夹实时神经元建模，以阐明突变的分子和神经元尺度表型后果。N1662D突变几乎完全阻止了快速失活而不影响活化。野生型和N1662D通道结构的比较表明，残基N1662和Q1494之间的双功能氢键形成对于快速失活至关重要。快速失活也可以防止工程Q1494A或Q1494LNav1.2通道变种，而Q1494E或Q1494K变异导致不完全失活和持续电流。分子动力学模拟显示疏水性IFM-基序相对于野生型具有N1662D和Q1494L变体对其受体位点的亲和力降低。这些结果表明，N1662和Q1494之间的相互作用支持失活门的稳定性和取向，并且对于快速失活的发展至关重要。六个DEE相关的Nav1.2变体，还评估了映射到已知与快速失活有关的通道片段的突变。值得注意的是,L1657P变体还阻止了快速失活，并产生了与N1662D相似的生物物理特征，而M1501V，M1501T,F1651C,P1658S,和A1659V变体产生的生物物理特性与动态动作电位钳制实验中混合神经元的功能获得和增强的动作电位放电一致。矛盾的是,低密度N1662D或L1657P电流增强动作电位激发，而密度增加导致持续的去极化。我们的结果为Nav1.2通道快速失活的分子机制提供了新的结构见解，并为SCN2A相关EI-DEE的治疗策略提供了信息。非失活Nav1.2通道对神经元兴奋性的贡献可能构成SCN2A相关DEE发病机理中的独特细胞机制。
