Phosphorylation-dependent regulation of calcium channels by macromolecular complexes

大分子复合物对钙通道的磷酸化依赖性调节

基本信息

  • 批准号:
    9979954
  • 负责人:
  • 金额:
    $ 73.02万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2019
  • 资助国家:
    美国
  • 起止时间:
    2019-08-01 至 2023-05-31
  • 项目状态:
    已结题

项目摘要

Our overall goal is to discover details of fundamental mechanisms underlying regulation of CaV1.2 channels that have eluded more than four decades of investigation. We propose to use novel tools and approaches to identify novel proteins, supramolecular complexes, and signaling pathways affecting CaV1.2 channels as the basis for targeted drug development for arrhythmias. Although it is well-established that phosphorylation by cyclic AMP (cAMP)-PKA, but not Ca2+/calmodulin kinase II (CaMKII), is the fundamental process by which b- adrenergic stimulation controls Ca2+ influx via CaV1.2 in the heart, the molecular targets of PKA remain unknown. A detailed molecular understanding of CaV1.2 regulation in myocytes has been hampered by the inability to recapitulate and then dissect in heterologous expression systems key aspects of CaV1.2 function in myocytes. Our novel tools surmount major obstacles that have limited progress in the field, and allow us to identify the neighboring proteome of CaV1.2 in the heart and probe molecular aspects of CaV1.2 regulation, using biochemical and electrophysiological techniques, within the context of cardiomyocytes, but with the power of a heterologous expression system. The failure thus far to identify any site as essential for adrenergic modulation led us to propose an alternative hypothesis: that a combination of phosphorylation sites in a1C is required for b-adrenergic stimulation of CaV1.2. To address this hypothesis, we generated mice with alanine- substitutions in rabbit a1C of all conserved and non-conserved consensus PKA phosphorylation sites (“35- mutant a1C”), and found that b-adrenergic regulation was not dependent upon any of these serine or threonine residues. Using a similar transgenic approach, we found that b-adrenergic regulation does not require phosphorylation of any of the 18 N-terminal, HOOK and GK domain consensus PKA phosphorylation sites in the b2b subunit. The next step is to create transgenic mice expressing b2b subunits with all PKA consensus sites removed (“33-mutant b2b”) and test regulation in a b2 knockout background. Thereafter, we will determine whether phosphorylation of either a1C or b subunits is sufficient to enable b-adrenergic regulation by crossing the 35-mutant a1C and the 33-mutant b2b mice. If adrenergic regulation is preserved, these results would shift a four-decade paradigm: the core CaV1.2 subunits are not the required PKA targets. Other aims of the proposal are to determine whether b-adrenergic stimulation of CaV1.2 is dependent upon a target extrinsic to CaV1.2 core subunits, and whether specifically attenuating b-adrenergic-modulation of CaV1.2 can suppress arrhythmogenesis. The feasibility of this approach is supported by the demonstration that disrupting the b-a interaction prevents b-adrenergic regulation of CaV1.2. The three Aims, which will provide key new understandings concerning the regulation of Ca2+ influx in cardiomyocytes, are highly relevant towards understanding cardiac pathologies and the molecular mechanisms responsible for the modulation of cardiac contractility.
我们的总体目标是发现CAV1.2渠道基本机制的基本机制的细节 这已经超过四十年的投资。我们建议使用新颖的工具和方法 识别新型蛋白质,超分子复合物和影响CAV1.2通道的信号通路为 心律不齐的靶向药物开发的基础。尽管良好地证明了磷酸化 环状AMP(CAMP)-PKA,但不是Ca2+/钙调蛋白激酶II(CAMKII),是B-的基本过程 肾上腺刺激控制Ca2+通过心脏中的Cav1.2影响Ca2+影响,PKA的分子靶标仍然存在 未知。对肌细胞中CAV1.2调节的详细分子理解已受到 无法概括并在异源表达系统中进行剖析,Cav1.2功能在 心肌细胞。我们的新颖工具克服了该领域进度有限的主要障碍,并让我们能够 确定Cav1.2的相邻蛋白质组和CAV1.2调节的探针分子方面, 在心肌细胞的背景下,使用生化和电生理技术 异源表达系统的功能。到目前为止,无法将任何站点识别为肾上腺素必不可少的 调节导致我们提出了另一种假设:A1C中磷酸化位点的组合是 B-肾上腺素能模拟的CAV1.2所需。为了解决这一假设,我们用丙氨酸产生了小鼠 在所有保守和未经保存的共识PKA磷酸化位点的兔A1c中取代(“ 35--) 突变A1c”),发现B-肾上腺素能调节不依赖于这些系列或苏氨酸 残留物。使用类似的转基因方法,我们发现B-肾上腺素能调节不需要 18个N末端,钩和GK结构域共有PKA磷酸化位点的磷酸化 B2B亚基。下一步是创建具有所有PKA共识的B2B亚基的转基因小鼠 在B2敲除背景下删除了站点(“ 33-突变B2B”)和测试调节。此后,我们将确定 A1C或B亚基的磷酸化是否足以通过跨越B-肾上腺素能调节 35个突变的A1C和33-突变的B2B小鼠。如果保留了肾上腺素调节,这些结果将改变 四十年范式:核心CAV1.2亚基不是所需的PKA目标。提案的其他目标 确定Cav1.2的B-肾上腺素能模拟是否取决于靶外cav1.2。 核心亚基,以及是否特别衰减CAV1.2的B-肾上腺素能调节可以抑制 心律失常。这种方法的可行性得到了破坏B-A的演示 相互作用阻止了CAV1.2的B-肾上腺素能调节。这三个目标将提供新的新目标 关于在心肌细胞中CA2+影响调节的理解,与 了解心脏病理和导致心脏调节的分子机制 收缩力。

项目成果

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专著数量(0)
科研奖励数量(0)
会议论文数量(0)
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数据更新时间:2024-06-01

Steven O Marx的其他基金

Roles of Rad and other CaV1.2 neighboring proteins in regulating cardiac function in health and disease
Rad 和其他 CaV1.2 邻近蛋白在健康和疾病中调节心脏功能中的作用
  • 批准号:
    10628915
    10628915
  • 财政年份:
    2023
  • 资助金额:
    $ 73.02万
    $ 73.02万
  • 项目类别:
Investigating Cardiac Ion Channels by Novel Methods
通过新方法研究心脏离子通道
  • 批准号:
    10219521
    10219521
  • 财政年份:
    2021
  • 资助金额:
    $ 73.02万
    $ 73.02万
  • 项目类别:
Dynamic changes of the Nav1.5 interactome and contributions to heart failure
Nav1.5 相互作用组的动态变化及其对心力衰竭的影响
  • 批准号:
    10478131
    10478131
  • 财政年份:
    2021
  • 资助金额:
    $ 73.02万
    $ 73.02万
  • 项目类别:
Investigating Cardiac Ion Channels by Novel Methods
通过新方法研究心脏离子通道
  • 批准号:
    10418713
    10418713
  • 财政年份:
    2021
  • 资助金额:
    $ 73.02万
    $ 73.02万
  • 项目类别:
Dynamic changes of the Nav1.5 interactome and contributions to heart failure
Nav1.5 相互作用组的动态变化及其对心力衰竭的影响
  • 批准号:
    10317712
    10317712
  • 财政年份:
    2021
  • 资助金额:
    $ 73.02万
    $ 73.02万
  • 项目类别:
Investigating Cardiac Ion Channels by Novel Methods
通过新方法研究心脏离子通道
  • 批准号:
    10673191
    10673191
  • 财政年份:
    2021
  • 资助金额:
    $ 73.02万
    $ 73.02万
  • 项目类别:
Dynamic changes of the Nav1.5 interactome and contributions to heart failure
Nav1.5 相互作用组的动态变化及其对心力衰竭的影响
  • 批准号:
    10658902
    10658902
  • 财政年份:
    2021
  • 资助金额:
    $ 73.02万
    $ 73.02万
  • 项目类别:
Phosphorylation-dependent regulation of calcium channels by macromolecular complexes
大分子复合物对钙通道的磷酸化依赖性调节
  • 批准号:
    10161818
    10161818
  • 财政年份:
    2019
  • 资助金额:
    $ 73.02万
    $ 73.02万
  • 项目类别:
Phosphorylation-dependent regulation of calcium channels by macromolecular complexes
大分子复合物对钙通道的磷酸化依赖性调节
  • 批准号:
    10425277
    10425277
  • 财政年份:
    2019
  • 资助金额:
    $ 73.02万
    $ 73.02万
  • 项目类别:
Calmodulin regulation of Na+ channels in neurons and cardiomyocytes
钙调蛋白对神经元和心肌细胞Na通道的调节
  • 批准号:
    8965516
    8965516
  • 财政年份:
    2014
  • 资助金额:
    $ 73.02万
    $ 73.02万
  • 项目类别:

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Phosphorylation-dependent regulation of calcium channels by macromolecular complexes
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