Structural consequences of PKC-dependent phosphorylation of Kv7.2

Kv7.2 PKC 依赖性磷酸化的结构后果

• 批准号: 10609077
• 负责人: Crystal Rae Archer
• 金额: $ 12.5万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10609077
• 关键词: 3-Dimensional; Affect; Affinity; Award; Binding; Binding Sites; Biological Assay; Ca(2+)-Calmodulin Dependent Protein Kinase; Calmodulin; Career Mobility; Cell Culture Techniques; Cryoelectron Microscopy; Data; Disease; Electrophysiology (science); Encephalopathies; Environment; Epilepsy; Event; Financial Support; Foundations; Functional disorder; Future; G-Protein-Coupled Receptors; GTP-Binding Proteins; Health; Human; Interdisciplinary Study; Ion Channel; Isotope Labeling; Maps; Molecular; Muscarinic M1 Receptor; Mutation; Neonatal; Pain; Pharmacology; Phase; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphorylation; Physiology; Play; Protein Isoforms; Protein Kinase; Protein Kinase C; Regulation; Reporting; Research; Resources; Role; Seizures; Serine; Signal Transduction; Signaling Molecule; Site; Stroke; Structure; Techniques; Testing; Training; Traumatic Brain Injury; X-Ray Crystallography; Xenopus; alpha helix; biophysical techniques; career; cofactor; improved; innovation; mutant; neuronal excitability; novel; optogenetics; prevent; programs; stoichiometry; structural biology; supportive environment

Project Summary M channels are critical for regulating the excitability of neurons. Dysfunction of M channel activity can cause epilepsy. While M channels have been intensely studied, the interplay of several G-protein-regulated signaling cofactors on these channels is still poorly understood. M channels are hetero-tetrameric pore structures formed by the combination of subunits Kv7.2-5. Over 80 mutations have been mapped to the Kv7.2 subunit, being a primary cause of neonatal epilepsy. Many of these mutations lie within the binding domains for at least three critical signaling cofactors: calmodulin (CaM), phosphatidylinositol 4,5-bisphosphate (PIP2) and protein kinase (PKC). How PIP2 and CaM binding to Kv7.2 harmonize to fine tune channel activity is obscure. Moreover, the role played by PKC in tuning this binding is obscure. My preliminary data shows that PIP2 and CaM may simultaneously bind the B helix with phosphorylation tempering this binding. My NMR studies so far show that phosphorylation reconfigures the apoCaM-B helix interaction, suggesting the interplay between these cofactors has significant impact on channel structure. I will test the overarching hypothesis that phosphorylation at S520 and S527 fine-tunes the ability of PIP2 and CaM to bind to Kv7.2 and control M channel activity. This hypothesis will be tested using three aims and will provide mechanistic understanding of how phosphorylation, and dependent PIP2 and CaM binding affects the structure of Kv7.2 to control channel activity. In Aim 1, I will use advanced 3D and 4D NMR to resolve the solution structure of purified Kv7.2 C terminus. Aim 2 will use these NMR spectra to define the affinity of PIP2 to Kv7.2 and describe the stoichiometry and mode of binding between PIP2 and the multiple sites on Kv7.2. Aim 3 will elaborate how phosphorylation within the B helix directs the interplay between CaM and PIP2 binding to Kv7.2. The proposed study is innovative because it will address a longstanding question about how and where PIP2 binds Kv7.2, and will elaborate how phosphorylation directs the interplay between CaM and PIP2 as they bind Kv7.2. My training in advanced 3D and 4D NMR will be critical for my career advancement as I plan to use this rigorous technique throughout my career. The rich resources and supportive environment at UT Health combined with my expertise in biophysical methods on ion channels makes me the ideal candidate to study the mechanisms underlying the regulation of M channel activity. The MOSAIC career award will provide valuable financial support to help me begin my multidisciplinary research program focused on elucidating the molecular mechanisms of ion channel regulation.

项目摘要 M通道是调节神经元兴奋性的关键。M通道活动功能障碍可 导致癫痫。虽然M通道已经得到了深入的研究，但几种G蛋白调节的相互作用 这些通道上的信号辅助因子仍然知之甚少。M孔道为异四聚体孔道 Kv7.2-5亚基组合形成的结构。已有80多个突变被映射到Kv7.2 亚单位，是新生儿癫痫的主要原因。这些突变中的许多都存在于 至少三个关键的信号辅助因子：钙调蛋白(CaM)、磷脂酰肌醇4，5-二磷酸(PIP2)和 蛋白激酶(PKC)。PIP2和CaM与Kv7.2的结合如何协调以微调通道活动尚不清楚。 此外，PKC在调节这种结合方面所起的作用还不清楚。我的初步数据显示，PIP2和 CaM可以同时与B螺旋结合，并通过磷酸化来缓和这种结合。到目前为止我的核磁共振研究 表明磷酸化重新配置apoCaM-B螺旋相互作用，提示 这些共同因素对渠道结构有显著影响。我将测试最重要的假设 S520和S527的磷酸化微调PIP2和CaM结合Kv7.2和控制M的能力 通道活动。这一假设将通过三个目标进行检验，并将提供机械性的理解 以及依赖PIP2和CaM结合如何影响Kv7.2的结构以控制通道 活动。在目标1中，我将使用先进的3D和4D核磁共振来解析纯化的Kv7.2 C的溶液结构 终点站。AIM 2将使用这些核磁共振谱来定义PIP2对Kv7.2的亲和力，并描述 化学计量学和PIP2与Kv7.2上多个位点的结合方式。目标3将详细说明如何 B螺旋内的磷酸化引导CaM和PIP2与Kv7.2结合的相互作用。建议数 这项研究具有创新性，因为它将解决一个长期存在的问题，即PIP2如何以及在哪里绑定Kv7.2， 并将详细阐述磷酸化如何指导CaM和PIP2之间的相互作用，因为它们结合Kv7.2。我的 高级3D和4D核磁共振方面的培训对我的职业发展至关重要，因为我计划使用这一严格的 技术贯穿了我的职业生涯。UT Health丰富的资源和支持环境与 我在离子通道的生物物理方法方面的专业知识使我成为研究这种机制的理想人选。 这是M通道活动调节的基础。马赛克职业奖将提供宝贵的资金 支持我开始我的多学科研究计划，重点是阐明分子 离子通道调节机制。