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通道对于调节神经元的兴奋性至关重要。 M通道活动的功能障碍可以 引起癫痫。尽管对M通道进行了深入研究，但几个G蛋白调节的相互作用 这些通道上的信号传导辅因子仍然很少了解。 M通道是异光孔 亚基KV7.2-5组合形成的结构。超过80个突变已映射到KV7.2 亚基，是新生儿癫痫的主要原因。这些突变中有许多位于结合域内 至少三个关键信号辅助因子：钙调蛋白（CAM），磷脂酰肌醇4,5-双磷酸（PIP2）和 蛋白激酶（PKC）。 PIP2和CAM与KV7.2与微调通道活性的协调如何晦涩难懂。 此外，PKC在调整这种绑定中所扮演的角色是晦涩的。我的初步数据表明PIP2和 CAM可以同时通过这种结合来结合B螺旋的磷酸化回火。到目前为止我的NMR研究 表明磷酸化重新配置了apocam-b螺旋相互作用，这表明 这些辅助因子对信道结构有重大影响。我将测试总体假设 S520和S527微型磷酸化的PIP2和CAM与KV7.2并控制M的能力 通道活动。该假设将使用三个目标进行检验，并将提供机械理解 磷酸化以及依赖性PIP2和CAM结合的如何影响KV7.2控制通道的结构 活动。在AIM 1中，我将使用高级3D和4D NMR来解决纯化的KV7.2 C的溶液结构 终点。 AIM 2将使用这些NMR光谱来定义PIP2对KV7.2的亲和力并描述 pip2和kv7.2上多个位点之间的化学计量和结合模式。 AIM 3将详细说明如何 B螺旋中的磷酸化指导CAM与PIP2与KV7.2结合之间的相互作用。提议 研究具有创新性，因为它将解决一个长期存在的问题，即PIP2如何和位置绑定Kv7.2， 并将详细说明磷酸化如何在结合Kv7.2时指导CAM和PIP2之间的相互作用。我的 高级3D和4D NMR的培训对于我的职业发展至关重要，因为我计划使用这种严格 整个职业生涯。 UT Health的丰富资源和支持环境与 我在离子通道上生物物理方法方面的专业知识使我成为研究机制的理想候选者 M通道活性调节的基础。马赛克职业奖将提供宝贵的财务 支持我开始我的多学科研究计划，旨在阐明分子 离子通道调节的机理。