Calmodulin/Ca channel physiology in heart

心脏钙调蛋白/Ca 通道生理学

• 批准号: 6767491
• 负责人: DAVID T YUE
• 金额: $ 40.88万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6767491
• 关键词: Adenoviridae; action potentials; cAMP response element binding protein; calcium channel; calcium flux; calmodulin; cardiac myocytes; dihydropyridines; electrophysiology; fluorescence resonance energy transfer; guinea pigs; heart function; heart ventricle; laboratory rat; protein engineering; protein structure function; protein transport; recombinant proteins; tissue /cell culture; transfection /expression vector; voltage gated channel

The overall thrust is to deploy new molecular constructs---many inspired from channel mechanistic studies-for the discovery of fundamental, newly accessible arenas of CaM/Ca channel physiology in heart. This thrust drives three aims, addressing successively more general realms of cardiac physiology, each with fundamental and therapeutic implications. (1) To clarify facilitation of cardiac L-type Ca channels by Ca2+/CaM. By contrast to CDI, a distinct process of facilitated channel opening by Ca2+ (CDF) remains mysterious, despite its probable role in strengthening the heartbeat at faster heart rates. Still unclear is the actual strength of CDF in heart, and whether CDF shares rich CaM signaling features found in model experimental systems. Those systems permit study of engineered recombinant L-type channels that lack CDI and thereby permit maximal resolution of CDF. By contrast, incomplete separation of CDF from CDI seriously complicates study in heart. We will thus express engineered L-type channels (lacking CDI and dihydropyridine block) in myocytes. During dihydropyridine block of native channels, selective resolution of recombinant channels will permit unambiguous assessment and mechanistic dissection of CDF in the native setting. (2) To define the capabilities of cardiac L-type Ca channels to activate nuclear CREB. Such Ca2+ signaling appears crucial to the dynamic regulation of cardiac genes. In neurons, CaM not only regulates the channel to which it is bound, such CaM may also bridge preferential signaling of L-type channels to CREB. Here, we will define basic aspects of CREB signaling in myocytes, using distinctive methodologies such as CaM/L-type channel fusions to test whether the very CaM that modulates a channel is essential for triggering CREB. Optical FRET-based sensors of CREB activation also promise rapid temporal correlation of Ca2+ entry patterns and CREB activation. (3) To estimate the concentration of local endogenous CaM near L-type channels in heart cells. As CaMs responsive to local Ca2+ influx through L-type channels may be the initiatory Ca2+ sensors that ultimately trigger CREB and other nuclear factors, the number of CaMs privy to the local Ca2+ signal from channels is key to downstream signaling strength. Here, we will utilize CaM/L-type channel fusions, with polymer chain theory, to estimate the local concentration of endogenous CaM near channels. Preliminary results hint at mM concentrations, suggesting that a 'school' of local CaMs resides near channels. Overall, this proposal will answer fundamental unknowns of CaM/Ca channel physiology in the heart.

总体主旨是部署新的分子结构（许多分子结构受到通道机制研究的启发），以发现心脏中 CaM/Ca 通道生理学的基本、新近领域。这一推动力驱动了三个目标，相继解决了心脏生理学的更一般领域，每个目标都具有基础和治疗意义。 (1) 阐明Ca2+/CaM对心脏L型Ca通道的促进作用。与 CDI 相比，Ca2+ (CDF) 促进通道开放的独特过程仍然神秘，尽管它可能在加快心率时加强心跳方面发挥作用。目前仍不清楚 CDF 在心脏中的实际强度，以及 CDF 是否具有模型实验系统中丰富的 CaM 信号传导特征。这些系统允许研究缺乏 CDI 的工程重组 L 型通道，从而允许 CDF 的最大分辨率。相比之下，CDF 与 CDI 的不完全分离使心脏研究变得严重复杂化。因此，我们将在肌细胞中表达工程化的 L 型通道（缺乏 CDI 和二氢吡啶阻断）。在二氢吡啶阻断天然通道期间，重组通道的选择性解析将允许在天然环境中对 CDF 进行明确的评估和机械解剖。 (2)明确心脏L型Ca通道激活核CREB的能力。这种 Ca2+ 信号传导似乎对心脏基因的动态调节至关重要。在神经元中，CaM 不仅调节其所结合的通道，还可能将 L 型通道的优先信号传导桥接至 CREB。在这里，我们将定义肌细胞中 CREB ​​信号传导的基本方面，使用独特的方法（例如 CaM/L 型通道融合）来测试调节通道的 CaM 是否对于触发 CREB ​​至关重要。基于光学 FRET 的 CREB ​​激活传感器还可以实现 Ca2+ 进入模式和 CREB ​​激活的快速时间关联。 (3)估计心脏细胞L型通道附近局部内源性CaM的浓度。由于对通过 L 型通道的局部 Ca2+ 流入做出反应的 CaM 可能是最终触发 CREB ​​和其他核因子的起始 Ca2+ 传感器，因此对来自通道的局部 Ca2+ 信号敏感的 CaM 数量是下游信号强度的关键。在这里，我们将利用 CaM/L 型通道融合和聚合物链理论来估计通道附近内源性 CaM 的局部浓度。初步结果暗示了 mM 浓度，表明当地 CaM 的“学校”位于河道附近。总体而言，该提议将解答心脏 CaM/Ca 通道生理学的基本未知问题。