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:
    10161818
  • 负责人:
  • 金额:
    $ 72.97万
  • 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通道调节的基本机制的细节 四十多年来一直没有被调查我们建议使用新的工具和方法, 识别影响CaV1.2通道的新型蛋白质、超分子复合物和信号通路,作为 心律失常靶向药物开发的基础。尽管已经确定, 环AMP(cAMP)-PKA,而不是Ca 2 +/钙调蛋白激酶II(CaMKII),是B- 肾上腺素能刺激通过CaV1.2控制心脏中的Ca 2+内流,PKA的分子靶点仍然是 未知对心肌细胞中CaV1.2调节的详细分子理解受到以下因素的阻碍: 不能在异源表达系统中重现然后分解CaV1.2功能的关键方面, 肌细胞我们的新工具克服了限制该领域进展的主要障碍,并使我们能够 鉴定心脏中CaV1.2的邻近蛋白质组,并探测CaV1.2调节的分子方面, 使用生物化学和电生理技术,在心肌细胞的背景下,但与 异源表达系统的能力。到目前为止,未能确定任何部位是肾上腺素能神经递质的必需部位。 调节使我们提出了另一种假设:a1 C中磷酸化位点的组合是 CaV1.2的β-肾上腺素能刺激所需。为了证实这个假设,我们用丙氨酸- 所有保守和非保守共有PKA磷酸化位点在兔a1 C中的取代(“35- 37”)。 突变体a1 C”),并发现b-肾上腺素能调节不依赖于这些丝氨酸或苏氨酸中的任何一种 残基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 在一些实施方案中,本发明的化合物可以抑制18个N-末端、HOOK和GK结构域共有PKA磷酸化位点中的任何一个的磷酸化。 B2B亚基。下一步是建立转基因小鼠表达b2b亚基与所有PKA的共识 去除位点(“33-突变体B2B”)并在B2敲除背景下测试调节。此后,我们将确定 α 1 C或B亚基的磷酸化是否足以使β-肾上腺素能调节通过交叉 35个突变a1 C和33个突变b2b小鼠。如果肾上腺素能调节得以保留, 40年范式:核心CaV1.2亚基不是所需的PKA靶标。提案的其他目的 目的是确定CaV1.2的β-肾上腺素能刺激是否依赖于CaV1.2的外在靶点 核心亚基,以及特异性减弱CaV1.2的β-肾上腺素能调节是否可以抑制 胚胎发生这种方法的可行性得到了以下证据的支持: 相互作用阻止CaV1.2的β-肾上腺素能调节。这三个目标将提供关键的新 关于心肌细胞中Ca 2+内流调节的理解,与 了解心脏病理学和负责心脏调节的分子机制, 收缩性

项目成果

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Steven O Marx其他文献

Steven O Marx的其他文献

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{{ truncateString('Steven O Marx', 18)}}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
  • 财政年份:
    2023
  • 资助金额:
    $ 72.97万
  • 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
  • 财政年份:
    2021
  • 资助金额:
    $ 72.97万
  • 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
  • 财政年份:
    2021
  • 资助金额:
    $ 72.97万
  • 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
  • 财政年份:
    2021
  • 资助金额:
    $ 72.97万
  • 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
  • 财政年份:
    2021
  • 资助金额:
    $ 72.97万
  • 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
  • 财政年份:
    2021
  • 资助金额:
    $ 72.97万
  • 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
  • 财政年份:
    2021
  • 资助金额:
    $ 72.97万
  • 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
  • 财政年份:
    2019
  • 资助金额:
    $ 72.97万
  • 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:
    9979954
  • 财政年份:
    2019
  • 资助金额:
    $ 72.97万
  • 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
  • 财政年份:
    2014
  • 资助金额:
    $ 72.97万
  • 项目类别:

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