Dynamic changes in PIP2 binding sites and their impact on axonal targeting and function of epilepsy-associated KCNQ/Kv7 channels

PIP2 结合位点的动态变化及其对癫痫相关 KCNQ/Kv7 通道的轴突靶向和功能的影响

• 批准号: 10744934
• 负责人: Hee Jung Chung
• 金额: $ 38.05万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-15 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10744934
• 关键词: Action Potentials; Affect; Agonist; Anticonvulsants; Axon; Behavior; Behavioral; Binding; Binding Sites; Biochemistry; Biological Assay; Brain Diseases; Brain region; Calmodulin; Cell membrane; Charge; Chronic; Cognition; Cognitive; Complex; Coupling; Defect; Disease; Drug Targeting; Electrophysiology (science); Endocytosis; Endocytosis Inhibition; Endoplasmic Reticulum; Epilepsy; Etiology; Exocytosis; Foundations; Free Energy; Goals; Hippocampus; Image; Impairment; Knock-in Mouse; Knock-out; Knowledge; Lead; Lipids; Measurement; Mediating; Membrane; Membrane Lipids; Molecular; Mus; Mutation; Neurons; Pathogenicity; Pharmaceutical Preparations; Phosphatidylinositol 4,5-Diphosphate; Potassium; Potassium Channel; Publishing; Recurrence; Refractory; Regulation; Role; Seizures; Site; Stress; Surface; Testing; Variant; Work; cofactor; dominant genetic mutation; early onset; epileptic encephalopathies; interdisciplinary approach; mimetics; molecular dynamics; mutant; neuronal excitability; novel; trafficking; voltage

PROJECT SUMMARY Neuronal Kv7/KCNQ channels are homotetramers of Kv7.2 and heterotetramers of Kv7.2 and Kv7.3 that are highly expressed in the cortex and hippocampus, key brain regions for seizure, cognition and behavior. They produce voltage-dependent outward K+ current (IM) which potently suppresses neuronal excitability. Dominant mutations in Kv7.2 and Kv7.3 cause early-onset epileptic encephalopathy (EE) with severe cognitive and behavioral deficits, stressing a critical need to understand how EE variants dysregulate Kv7 channels. Our published studies show that Kv7 channels are preferentially enriched at the axonal plasma membrane via calmodulin (CaM) binding to intracellular helices A and B of Kv7.2, which mediates their trafficking from the endoplasmic reticulum to the axonal surface. Epilepsy variants in these helices reduce their axonal enrichment and seizures in mice, underscoring the key role of axonal Kv7 channels in excitability. Importantly, membrane lipid PIP2 is an essential cofactor for opening Kv7 channels as they are potently inhibited by its membrane depletion. However, the PIP2 binding residues that regulate neuronal Kv7 channels in different states (open or closed) and complex (homomers, heteromers, or CaM-bound) are unknown. Our recent work has revealed that the PIP2-binding residues in open Kv7.2 channels are different from those in closed state and CaM-bound open channels, and that select EE mutations of these sites induce both loss and gain of PIP2 sensitivity, and reduce their axonal enrichment. Thus, the PIP2-binding landscape is dynamic and may regulate both function and trafficking of Kv7 channels. The goals of this project are to identify (i) dynamic changes in PIP2 binding residues of neuronal Kv7 channels that control their axonal enrichment and function, (ii) mechanisms by which EE variants disrupt this modulation, and (iii) compounds that reverse this dysregulation. Our central hypothesis is that dynamic and coordinated binding of PIP2 and CaM regulates activation and trafficking of axonal Kv7 channels, whereas EE mutations increase neuronal excitability by impairing formation of this complex. To test this, the present project will execute 3 specific aims using interdisciplinary approach including molecular dynamic simulations, biochemistry, imaging, and electrophysiology. Aim 1 will identify PIP2 binding residues in CaM-bound and unbound Kv7 channels and test if their PIP2 binding and sensitivity are regulated by EE mutations, Kv7 agonists and PIP2 mimetic compounds. Aim 2 will identify how PIP2 binding modulates axonal surface enrichment of CaM-bound and unbound Kv7 channels by examining their exocytosis, endocytosis, and plasma membrane retention. Aim 3 will test if loss- and gain-of PIP2 modulations of axonal Kv7 channels lead to neuronal hyperexcitability in culture and conditional knock-in mice. In contrast to a well- established role of PIP2 in gating modulation of Kv7 channels, this project will provide novel concepts that their PIP2 binding sites change dynamically and modulate both function and trafficking of axonal Kv7 channels to impact IM and neuronal excitability, and reveal novel pathogenic mechanisms of EE variants in Kv7.2 and Kv7.3.

项目总结 神经元Kv7/KCNQ通道是Kv7.2的同质异构体，Kv7.2和Kv7.3的异构体是 在大脑皮质和海马区高度表达，这是癫痫发作、认知和行为的关键大脑区域。他们 产生电压依赖的外向钾电流(IM)，它有效地抑制神经元的兴奋性。占优势 Kv7.2和Kv7.3基因突变导致早发性癫痫脑病(EE)，伴有严重的认知和 行为缺陷，强调迫切需要了解EE变体是如何失调KV7通道的。我们的 已发表的研究表明，KV7通道优先在轴突质膜上通过 钙调素(CaM)与Kv7.2的细胞内螺旋A和B结合，介导它们从 内质网至轴突表面。这些螺旋结构中的癫痫变异减少了轴突的浓缩。 以及小鼠的癫痫发作，强调了轴突KV7通道在兴奋性中的关键作用。重要的是，膜 脂质PIP2是开放KV7通道的重要辅助因子，因为它们被其膜有效地抑制 耗尽。然而，调节神经元KV7通道的PIP2结合残基处于不同的状态(开放或 闭合)和络合物(同分异构体或CaM结合)是未知的。我们最近的工作揭示了 开放的Kv7.2通道中的PIP2结合残基不同于闭合状态和CaM结合的通道 开放的通道，并且这些位点的选择EE突变导致PIP2敏感性的丧失和获得，以及 减少他们的轴突浓缩。因此，PIP2结合的格局是动态的，并且可以调节这两种功能 和贩卖KV7频道。该项目的目标是确定(I)PIP2绑定的动态变化 控制其轴突丰富和功能的神经元KV7通道的残留物，(Ii)其机制 EE变异体破坏这种调节，以及(Iii)逆转这种失调的化合物。我们的中央 假设PIP2和CaM的动态和协调结合调节着PIP2和CaM的激活和运输 轴突KV7通道，而EE突变通过损害这种通道的形成而增加神经元的兴奋性 很复杂。为了测试这一点，本项目将使用跨学科方法执行3个具体目标 包括分子动力学模拟、生物化学、成像和电生理学。目标1将确定PIP2 在CaM结合和未结合的KV7通道中结合残基，并测试它们的PIP2结合和敏感性是否 受EE突变、KV7激动剂和PIP2模拟化合物的调控。AIM 2将确定PIP2如何结合 通过检测CaM结合和未结合的KV7通道的胞吐作用来调节轴突表面的浓缩， 内吞作用和质膜滞留。Aim 3将测试轴突PIP2调制的损失和增益 KV7通道导致培养和条件性敲入小鼠神经元过度兴奋。与井形成对比的是- PIP2在KV7通道的门控调制中的作用已经确立，这个项目将提供他们的新概念 PIP2结合位点动态变化并调节轴突KV7通道的功能和运输 影响IM和神经元兴奋性，并揭示Kv7.2和Kv7.3中EE变异的新致病机制。