Structural consequences of PKC-dependent phosphorylation of Kv7.2

项目摘要 M通道对于调节神经元的兴奋性至关重要。M通道活动功能障碍可 引起癫痫。虽然M通道已经被深入研究，但几种G蛋白调节的相互作用仍然存在。 这些通道上的信号传导辅因子仍然知之甚少。M通道是异源四聚体孔 由Kv7.2-5亚基组合形成的结构。超过80个突变已被映射到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位于 至少三种关键信号传导辅因子：钙调蛋白（CaM）、磷脂酰肌醇4，5-二磷酸（PIP 2）和 蛋白激酶（PKC）。PIP 2和CaM如何与Kv7.2结合协调以微调通道活性尚不清楚。 此外，PKC在调节这种结合中所起的作用还不清楚。我的初步数据显示，PIP 2和 钙调素可以同时结合B螺旋，磷酸化调节这种结合。到目前为止我的核磁共振研究 显示磷酸化重构了apoCaM-B螺旋相互作用，提示了 这些辅因子对通道结构具有显著影响。我将检验一个总体假设， 在S520和S527处的磷酸化精细调节PIP 2和CaM结合Kv7.2和对照M的能力。 渠道活动。这一假设将使用三个目标进行测试，并将提供机械的理解 磷酸化和依赖性PIP 2和CaM结合如何影响Kv7.2控制通道的结构 活动在目标1中，我将使用先进的三维和四维NMR来解析纯化的Kv 7.2 C的溶液结构 终点站目的2将使用这些NMR谱来定义PIP 2对Kv7.2的亲和力，并描述PIP 2与Kv7.2的亲和力。 PIP 2与Kv7.2上的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螺旋内的磷酸化引导CaM和PIP 2之间的相互作用结合Kv7.2。拟议 这项研究是创新性的，因为它将解决一个长期存在的问题，即PIP 2如何以及在何处结合Kv7.2， 并将详细阐述磷酸化如何指导钙调素和PIP 2之间的相互作用，因为他们结合Kv7.2。我 高级3D和4D NMR培训对我的职业发展至关重要，因为我计划使用这种严格的 技术贯穿我的职业生涯。UT Health的丰富资源和支持性环境与 我在离子通道生物物理学方法方面的专业知识使我成为研究 M通道活性的调节。MOSAIC职业奖将提供宝贵的财务支持， 支持，以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。

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.