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
基本信息
- 批准号:10744934
- 负责人:
- 金额:$ 38.05万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:Action PotentialsAffectAgonistAnticonvulsantsAxonBehaviorBehavioralBindingBinding SitesBiochemistryBiological AssayBrain DiseasesBrain regionCalmodulinCell membraneChargeChronicCognitionCognitiveComplexCouplingDefectDiseaseDrug TargetingElectrophysiology (science)EndocytosisEndocytosis InhibitionEndoplasmic ReticulumEpilepsyEtiologyExocytosisFoundationsFree EnergyGoalsHippocampusImageImpairmentKnock-in MouseKnock-outKnowledgeLeadLipidsMeasurementMediatingMembraneMembrane LipidsMolecularMusMutationNeuronsPathogenicityPharmaceutical PreparationsPhosphatidylinositol 4,5-DiphosphatePotassiumPotassium ChannelPublishingRecurrenceRefractoryRegulationRoleSeizuresSiteStressSurfaceTestingVariantWorkcofactordominant genetic mutationearly onsetepileptic encephalopathiesinterdisciplinary approachmimeticsmolecular dynamicsmutantneuronal excitabilitynoveltraffickingvoltage
项目摘要
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.
项目概要
神经元 Kv7/KCNQ 通道是 Kv7.2 的同四聚体以及 Kv7.2 和 Kv7.3 的异四聚体,
在大脑皮层和海马体中高度表达,这些区域是癫痫、认知和行为的关键大脑区域。他们
产生电压依赖性外向 K+ 电流 (IM),有效抑制神经元兴奋性。主导的
Kv7.2 和 Kv7.3 突变导致早发性癫痫性脑病 (EE),伴有严重的认知障碍和
行为缺陷,强调迫切需要了解 EE 变体如何失调 Kv7 通道。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,介导它们从
内质网至轴突表面。这些螺旋中的癫痫变异减少了它们的轴突富集
和小鼠癫痫发作,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 通道的重要辅助因子,因为它们被其膜有效抑制
消耗。然而,在不同状态(开放或开放)下调节神经元 Kv7 通道的 PIP2 结合残基
闭合)和复合物(同聚物、异聚物或 CaM 结合)未知。我们最近的工作揭示了
开放 Kv7.2 通道中的 PIP2 结合残基与关闭状态和 CaM 结合的残基不同
开放通道,并且这些位点的选择性 EE 突变会导致 PIP2 敏感性的丧失和增强,并且
减少轴突富集。因此,PIP2 结合景观是动态的,可以调节这两种功能
以及 Kv7 频道的贩运。该项目的目标是确定 (i) PIP2 结合的动态变化
神经元 Kv7 通道的残基控制其轴突富集和功能,(ii) 的机制
EE 变体破坏了这种调节,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激活和运输
轴突 Kv7 通道,而 EE 突变通过损害该通道的形成来增加神经元兴奋性
复杂的。为了测试这一点,当前项目将使用跨学科方法实现 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 将识别 PIP2
CaM 结合和未结合的 Kv7 通道中的结合残基,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 模拟化合物调节。目标 2 将确定 PIP2 的结合方式
通过检查胞吐作用来调节 CaM 结合和未结合 Kv7 通道的轴突表面富集,
内吞作用和质膜滞留。目标 3 将测试轴突 PIP2 调制是否丢失和增益
Kv7 通道导致培养小鼠和条件敲入小鼠的神经元过度兴奋。与良好相比
确立了 PIP2 在 Kv7 通道的门控调制中的作用,该项目将提供新的概念
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 Kv7.2 和 Kv7.3 中 EE 变异的新致病机制。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Hee Jung Chung其他文献
Hee Jung Chung的其他文献
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{{ truncateString('Hee Jung Chung', 18)}}的其他基金
Super-Resolution Fluorescence Microscopy of Synaptic Plasticity on Unmodified Brain Slices in Health and Tauopathy
健康和 Tau 病未修饰脑切片突触可塑性的超分辨率荧光显微镜
- 批准号:
10729062 - 财政年份:2023
- 资助金额:
$ 38.05万 - 项目类别:
Super-Resolution Microscopy of Neuronal Synapses with Small Quantum Dots and Advanced Imaging Tools
使用小量子点和先进成像工具对神经元突触进行超分辨率显微镜检查
- 批准号:
9384063 - 财政年份:2017
- 资助金额:
$ 38.05万 - 项目类别:
Super-Resolution Microscopy of Neuronal Synapses with Advanced Imaging Tools
使用先进成像工具对神经元突触进行超分辨率显微镜检查
- 批准号:
10299205 - 财政年份:2017
- 资助金额:
$ 38.05万 - 项目类别:
Super-Resolution Microscopy of Neuronal Synapses with Advanced Imaging Tools
使用先进成像工具对神经元突触进行超分辨率显微镜检查
- 批准号:
10684709 - 财政年份:2017
- 资助金额:
$ 38.05万 - 项目类别:
Super-Resolution Microscopy of Neuronal Synapses with Advanced Imaging Tools
使用先进成像工具对神经元突触进行超分辨率显微镜检查
- 批准号:
10467027 - 财政年份:2017
- 资助金额:
$ 38.05万 - 项目类别:
Super-Resolution Microscopy of Neuronal Synapses with Small Quantum Dots and Advanced Imaging Tools
使用小量子点和先进成像工具对神经元突触进行超分辨率显微镜检查
- 批准号:
9975253 - 财政年份:2017
- 资助金额:
$ 38.05万 - 项目类别:
Super-Resolution Microscopy of Small Quantum Dots to Elucidate the Mechanisms of Alzheimer's Disease
小量子点的超分辨率显微镜阐明阿尔茨海默病的机制
- 批准号:
9478382 - 财政年份:2016
- 资助金额:
$ 38.05万 - 项目类别:
Super-Resolution Microscopy of Small Quantum Dots to Elucidate the Mechanisms of Alzheimer's Disease
小量子点的超分辨率显微镜阐明阿尔茨海默病的机制
- 批准号:
9160604 - 财政年份:2016
- 资助金额:
$ 38.05万 - 项目类别:
Super-Resolution Microscopy of Small Quantum Dots to Elucidate the Mechanisms of Alzheimer's Disease
小量子点的超分辨率显微镜阐明阿尔茨海默病的机制
- 批准号:
9274105 - 财政年份:2016
- 资助金额:
$ 38.05万 - 项目类别:
Super-Resolution Microscopy of Small Quantum Dots to Elucidate the Mechanisms of Alzheimer's Disease
小量子点的超分辨率显微镜阐明阿尔茨海默病的机制
- 批准号:
9918990 - 财政年份:2016
- 资助金额:
$ 38.05万 - 项目类别:
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