Signaling Pathways and Regulators of Calcium Channels in Heart

心脏钙通道的信号通路和调节因子

• 批准号: 10063904
• 负责人: Daniel Dennis Roybal
• 金额: $ 3.78万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-12-20 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10063904
• 关键词: Action Potentials; Adrenergic Agents; Adrenergic beta-Agonists; Affect; Alanine; Arrhythmia; Biochemical; Biotin; Blood flow; C-terminal; Calcium; Calcium Channel; Cardiac; Cardiac Myocytes; Catecholamines; Cavia; Cells; Complex; Consensus; Coupling; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Dihydropyridines; Doxycycline; Epitopes; Exercise; Failure; Gene Expression; Generations; Goals; Heart; Human; Immunoprecipitation; In Situ; Knock-in Mouse; Knockout Mice; Label; Laboratories; Macromolecular Complexes; Mass Spectrum Analysis; Mediating; Membrane; Metabolic; Methods; Molecular; Mus; Mutate; N-terminal; Organ; Oryctolagus cuniculus; Pathologic; Peroxidases; Phase; Phosphorylation; Phosphorylation Site; Physiological; Process; Protein Kinase; Proteins; Rattus; Regulation; Regulatory Pathway; Resistance; RyR2; Ryanodine Receptor Calcium Release Channel; SH3 Domains; Sarcoplasmic Reticulum; Serine; Signal Pathway; Signal Transduction; Site; Structure; Sudden Death; Sympathetic Nervous System; System; Testing; Threonine; Tissues; Transgenic Mice; Up-Regulation; base; beta-adrenergic receptor; fighting; inducible gene expression; insight; knock-down; member; mutant; novel; preservation; protein activation; protein complex; reconstitution; response; small hairpin RNA; trafficking; voltage

In the heart, calcium influx via Cav1.2 channels has a key role in excitation-contraction coupling, and in determining the plateau phase of the action potential. Although CaV1.2 channels are known to associate with proteins that regulate channel trafficking, localization, turnover, and function, the components of these protein complexes have not yet been fully identified. Physiologic b-adrenergic activation of PKA during the sympathetic “fight or flight” response increases calcium influx through CaV1.2 in cardiomyocytes, leading to increased cardiac contractility. In pathological conditions, increased CaV1.2 currents can trigger electrical instability, early after- depolarizations, arrhythmias, and sudden death, frequently in the setting of adrenergic stimulation or decreased repolarizing currents. The molecular mechanisms of b-adrenergic regulation of CaV1.2 in cardiomyocytes are incompletely known, but up-regulation of CaV1.2 mediated by activation of PKA is required for this process. Based upon the failure to identify the regulatory sites in the heart, the difficulties in reconstituting the regulation using heterologous expression, and the challenges in creating knock-in mice or using adenoviral-based expression, the Marx laboratory developed the straightforward but rigorous approach of using doxycycline- inducible, tissue-specific, transgenic-mice-expressing FLAG-epitope-tagged, dihydropyridine (DHP)-resistant pore-forming a1C subunits. Recent data suggest that b-adrenergic regulation of CaV1.2 does not require any combination of potential PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse a1C subunits. b-adrenergic regulation of a1C may require, however, a unique combination of species-specific phospho-regulatory sites in a1C. To test this hypothesis, I generated transgenic mice with doxycycline-inducible expression of rabbit a1C with alanine-substitutions of all conserved and non-conserved potential PKA sites in the intracellular regions (N-terminal, intracellular loops, and C-terminal regions). If b-adrenergic regulation is preserved in these mice, I will cross these mice with mice expressing a mutant b2b subunit in which consensus PKA phosphorylation sites are substituted with alanines. This study will test whether prior failures to identify a mechanism is because of redundancy between the a and b subunits and will provide the definitive answer about whether PKA phosphorylation of any a1C or b2 residues is necessary. Since it appears that the a1C and b2 subunits may not the primary functional PKA targets and due to the inability to reconstitute adrenergic regulation when the primary subunits are heterologously expressed, I further hypothesize that additional proteins expressed in cardiomyocytes may be required for adrenergic regulation of CaV1.2. In Aim 2, I seek to identify and test these novel regulators of CaV1.2 in the heart. The two Aims will provide new insights the mechanisms responsible for b-adrenergic regulation of Ca2+ influx in cardiomyocytes.

在心脏中，通过 Cav1.2 通道的钙流入在兴奋-收缩耦合以及在 尽管已知 CaV1.2 通道与动作电位的平台期相关。 调节通道运输、定位、周转和功能的蛋白质，这些蛋白质的成分 交感神经期间PKA的生理性β-肾上腺素能激活复合物尚未完全确定。 “战斗或逃跑”反应通过 CaV1.2 增加心肌细胞中的钙流入，导致心肌细胞 在病理条件下，CaV1.2 电流增加会引发电不稳定，早期后遗症。 去极化、心律失常和猝死，通常发生在肾上腺素能刺激或减少的情况下 心肌细胞中 CaV1.2 的复极电流的分子机制是 尚不完全清楚，但该过程需要通过 PKA 激活介导的 CaV1.2 上调。 由于未能确定心脏的调节位点，重建调节的困难 使用异源表达，以及创建敲入小鼠或使用基于腺病毒的挑战 表达，马克思实验室开发了使用强力霉素的简单但严格的方法- 可诱导、组织特异性、转基因小鼠表达 FLAG 表位标记、二氢吡啶 (DHP) 抗性 最近的数据表明 CaV1.2 的 β 肾上腺素能调节不需要任何作用。 人、豚鼠、兔、大鼠和小鼠 a1C 中保守的潜在 PKA 磷酸化位点组合 然而，a1C 的 b-肾上腺素能调节可能需要物种特异性的独特组合。 为了验证这一假设，我培育了多西环素诱导的转基因小鼠。 兔 a1C 的表达，用丙氨酸取代了所有保守和非保守的潜在 PKA 位点 细胞内区域（N 末端、细胞内环和 C 末端区域）。 保存在这些小鼠中，我将这些小鼠与表达突变 b2b 亚基的小鼠杂交，其中一致 PKA 磷酸化位点被丙氨酸取代 本研究将测试先前未能识别的位点。 该机制是由于 a 和 b 亚基之间的冗余，并将提供关于 任何 a1C 或 b2 残基的 PKA 磷酸化是否是必要的，因为 a1C 和 b2 似乎是必需的。 亚基可能不是主要的功能性 PKA 靶标，并且由于无法重建肾上腺素能调节 当主要亚基异源表达时，我进一步追寻额外表达的蛋白质 心肌细胞中的 CaV1.2 可能需要肾上腺素调节。在目标 2 中，我试图识别并测试这些。 心脏中 CaV1.2 的新型调节因子将提供新的见解。 心肌细胞中 Ca2+ 流入的 b 肾上腺素调节。