Phosphorylation-dependent regulation of calcium channels by macromolecular complexes

我们的总体目标是发现调控CaV1.2通道的基本机制的细节 这是40多年来一直未能调查的问题。我们建议使用新的工具和方法来 确定影响CaV1.2通道的新蛋白质、超分子复合体和信号通路是 为治疗心律失常的靶向药物开发奠定了基础。尽管众所周知，磷酸化是通过 CAMP(CAMP)-PKA，而不是钙/钙调蛋白激酶II(CaMKII)是b-AMP(CAMP)-PKA的基本过程。 肾上腺素能刺激通过CaV1.2在心脏控制钙内流，PKA的分子靶点仍然存在 未知。对CaV1.2在心肌细胞中调节的详细分子理解受到了阻碍 不能概括和剖析异源表达系统中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CaV1.2在心脏中的邻近蛋白质组并探索CaV1.2调控的分子方面， 使用生化和电生理技术，在心肌细胞的背景下，但与 异源表达系统的力量。迄今未能将任何部位确定为肾上腺素能必需的部位 调节机制使我们提出了另一种假设：A1C中的磷酸化位点的组合是 刺激CaV1.2的b-肾上腺素能所需。为了解决这一假设，我们产生了丙氨酸- 兔A1C中所有保守和非保守的一致的PKA磷酸化位点(“35- 突变体A1C“)，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个 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、钩子和GK结构域共有的PKA磷酸化位点中的任何一个的磷酸化 B2B亚单位。下一步是创造表达所有PKA共识的B2B亚基的转基因小鼠 在b2基因敲除背景中删除位点(“33-突变型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交叉来实现b-肾上腺素能调节 35个突变的A1C和33个突变的B2B小鼠。如果肾上腺素能调节得到保护，这些结果将改变 40年范式：核心CaV1.2亚单位不是必需的PKA目标。该提案的其他目的 确定CaV1.2的b-肾上腺素能刺激是否依赖于CaV1.2的外在靶点 核心亚单位，以及特异性减弱CaV1.2的b-肾上腺素能调制是否能抑制 心律失常。这种方法的可行性得到了扰乱b-a的演示的支持 相互作用阻止了CaV1.2的b-肾上腺素能调节。三个目标，这将提供关键的新 对心肌细胞内钙内流调节的认识，与 了解心脏病理和心脏调节的分子机制 伸缩性。

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.