Structural consequences of PKC-dependent phosphorylation of Kv7.2
Kv7.2 PKC 依赖性磷酸化的结构后果
基本信息
- 批准号:10609077
- 负责人:
- 金额:$ 12.5万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:3-DimensionalAffectAffinityAwardBindingBinding SitesBiological AssayCa(2+)-Calmodulin Dependent Protein KinaseCalmodulinCareer MobilityCell Culture TechniquesCryoelectron MicroscopyDataDiseaseElectrophysiology (science)EncephalopathiesEnvironmentEpilepsyEventFinancial SupportFoundationsFunctional disorderFutureG-Protein-Coupled ReceptorsGTP-Binding ProteinsHealthHumanInterdisciplinary StudyIon ChannelIsotope LabelingMapsMolecularMuscarinic M1 ReceptorMutationNeonatalPainPharmacologyPhasePhosphatidylinositol 4,5-DiphosphatePhosphatidylinositolsPhosphorylationPhysiologyPlayProtein IsoformsProtein KinaseProtein Kinase CRegulationReportingResearchResourcesRoleSeizuresSerineSignal TransductionSignaling MoleculeSiteStrokeStructureTechniquesTestingTrainingTraumatic Brain InjuryX-Ray CrystallographyXenopusalpha helixbiophysical techniquescareercofactorimprovedinnovationmutantneuronal excitabilitynoveloptogeneticspreventprogramsstoichiometrystructural biologysupportive environment
项目摘要
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.
项目概要
M 通道对于调节神经元的兴奋性至关重要。 M 通道活性功能障碍可
引起癫痫。虽然 M 通道已被深入研究,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通道是异四聚体孔
由亚基Kv7.2-5组合形成的结构。 Kv7.2 已映射超过 80 个突变
亚单位,是新生儿癫痫的主要原因。许多这些突变位于结合域内
至少三种关键信号辅助因子: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 结合如何协调微调通道活动尚不清楚。
此外,PKC 在调节这种结合中所起的作用尚不清楚。我的初步数据显示 PIP2 和
CaM 可能同时结合 B 螺旋,磷酸化会缓和这种结合。到目前为止我的核磁共振研究
显示磷酸化重新配置了 apoCaM-B 螺旋相互作用,表明两者之间的相互作用
这些辅助因素对通道结构有重大影响。我将测试总体假设
S520 和 S527 的磷酸化微调 PIP2 和 CaM 结合 Kv7.2 和控制 M 的能力
渠道活动。该假设将使用三个目标进行测试,并将提供机械理解
磷酸化以及依赖性 PIP2 和 CaM 结合如何影响 Kv7.2 控制通道的结构
活动。在目标 1 中,我将使用先进的 3D 和 4D NMR 来解析纯化的 Kv7.2 C 的溶液结构
终点站。目标 2 将使用这些 NMR 谱来定义 PIP2 与 Kv7.2 的亲和力并描述
PIP2 与 Kv7.2 上多个位点之间的化学计量和结合模式。目标 3 将详细说明如何
B 螺旋内的磷酸化指导 CaM 和 PIP2 与 Kv7.2 结合之间的相互作用。拟议的
研究具有创新性,因为它将解决一个长期存在的问题:PIP2 如何以及在何处结合 Kv7.2,
并将详细阐述磷酸化如何指导 CaM 和 PIP2 结合 Kv7.2 时的相互作用。我的
高级 3D 和 4D NMR 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的方法
技术贯穿我的整个职业生涯。 UT Health 丰富的资源和支持环境与
我在离子通道生物物理方法方面的专业知识使我成为研究其机制的理想人选
M 通道活动的调节的基础。 MOSAIC 职业奖将提供宝贵的财务支持
支持帮助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。
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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Crystal Rae Archer其他文献
Crystal Rae Archer的其他文献
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{{ truncateString('Crystal Rae Archer', 18)}}的其他基金
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:
10429142 - 财政年份:2022
- 资助金额:
$ 12.5万 - 项目类别:
Biophysical interactions of PIP2 and calmodulin with KCNQ (Kv7) K+ ion channels
PIP2 和钙调蛋白与 KCNQ (Kv7) K 离子通道的生物物理相互作用
- 批准号:
8838438 - 财政年份:2015
- 资助金额:
$ 12.5万 - 项目类别:
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