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.

项目摘要 神经元KV7/KCNQ频道是KV7.2的同型元素，而KV7.2和KV7.3的异光聚体是 高度表达在皮层和海马，癫痫发作，认知和行为的关键大脑区域。他们 产生依赖电压的向外K+电流（IM），可有效抑制神经元兴奋性。主导的 KV7.2和KV7.3的突变引起的早期发作性癫痫性脑病（EE）具有严重的认知和 行为缺陷，强调了解EE变体如何失调KV7通道的迫切需要。我们的 已发表的研究表明，KV7通道优先富集在轴突质膜上 钙调蛋白（CAM）与KV7.2的细胞内螺旋a和B结合，从 内质网向轴突表面。这些螺旋中的癫痫变量减少了轴突富集 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是打开KV7通道的必不可少的辅助因子，因为它们被膜有效抑制 消耗。但是，在不同状态下调节神经元KV7通道的PIP2结合残基（开放或 封闭）和复杂（同源物，异构体或cam结合）是未知的。我们最近的工作揭示了 开放KV7.2通道中的PIP2结合残基与封闭状态和凸轮的pip 2结合残基不同 开放通道，并且选择这些位点的EE突变会引起PIP2敏感性的损失和增益，并且 减少轴突富集。因此，PIP2结合景观是动态的，并且可能调节两个功能 和贩运KV7渠道。该项目的目标是确定（i）PIP2绑定的动态变化 控制其轴突富集和功能的神经元KV7通道的残基，（ii） EE变体破坏了这种调制，（iii）化合物会扭转这种失调。我们的中心 假设是PIP2和CAM的动态和协调结合可以调节激活和运输 轴突KV7通道，而EE突变通过损害形成来增加神经元兴奋性 复杂的。为了测试这一点，本项目将使用跨学科方法执行3个特定目标 包括分子动态模拟，生物化学，成像和电生理学。 AIM 1将识别PIP2 在CAM结合和未结合的KV7通道中结合残基，并测试其PIP2结合和灵敏度是否为 由EE突变，KV7激动剂和PIP2模拟化合物调节。 AIM 2将确定pip2的绑定 通过检查其胞吐作用，调节CAM结合和未结合的KV7通道的轴突表面富集， 内吞作用和质膜保留。 AIM 3将测试轴突的损失和损失2调制 KV7通道导致培养物和条件敲门小鼠的神经元过度兴奋性。与一个好的相反 PIP2在KV7渠道的门控调制中的确定作用，该项目将提供新颖的概念 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，并揭示了KV7.2和KV7.3中EE变体的新型致病机制。