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

项目摘要 神经元Kv 7/KCNQ通道是Kv7.2的同源四聚体和Kv7.2和Kv7.3的异源四聚体， 在大脑皮层和海马高度表达，这是癫痫发作、认知和行为的关键大脑区域。他们 产生电压依赖性外向K+电流（IM），其有效抑制神经元兴奋性。主导 Kv7.2和Kv7.3的突变导致早发性癫痫性脑病（EE），伴有严重的认知和 行为缺陷，强调迫切需要了解EE变体如何失调Kv 7通道。我们 已发表的研究表明，Kv 7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，介导它们从细胞内运输。 内质网到轴突表面。这些螺旋中的癫痫变体减少了它们的轴突富集 和小鼠癫痫发作，强调了轴突Kv 7通道在兴奋性中的关键作用。重要的是，膜 脂质PIP 2是打开Kv 7通道的必要辅因子，因为它们被其膜有效抑制 耗尽然而，在不同状态下（开放或开放）调节神经元Kv 7通道的PIP 2结合残基， 封闭的）和复杂的（同聚体、异聚体或CaM结合的）尚不清楚。我们最近的研究显示 开放的Kv7.2通道中的PIP 2结合残基不同于关闭状态和CaM结合的那些， 开放通道，并且这些位点的选择EE突变诱导PIP 2敏感性的丧失和获得，以及 减少它们的轴突富集。因此，PIP 2结合景观是动态的，并且可以调节这两种功能 以及Kv 7频道的非法交易本项目的目标是确定（i）PIP 2绑定的动态变化 控制其轴突富集和功能的神经元Kv 7通道的残基，（ii） EE变体破坏这种调节，和（iii）逆转这种失调的化合物。我们的中央 假设是PIP 2和CaM的动态和协调结合调节细胞的激活和运输 轴突Kv 7通道，而EE突变增加神经元的兴奋性，通过损害这种形成， 复杂.为了验证这一点，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将识别PIP 2 结合CaM结合和未结合的Kv 7通道中的结合残基，并测试它们的PIP 2结合和灵敏度是否 受EE突变、Kv 7激动剂和PIP 2模拟化合物调节。目标2将确定PIP 2如何结合 通过检查它们的胞吐作用调节CaM结合和未结合Kv 7通道的轴突表面富集， 内吞作用和质膜滞留。目标3将测试轴突的PIP 2调制的损失和获得 Kv 7通道导致培养和条件敲入小鼠中的神经元过度兴奋。与之相对的是- PIP 2在Kv 7通道门控调制中的作用，该项目将提供新的概念， PIP 2结合位点动态变化并调节轴突Kv 7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变体的新致病机制。

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.