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.

总体推力是部署新的分子构建体 - 许多人灵感来自渠道的机械研究，从而发现了心脏中CAM/CA通道生理的基本，新近可进入的领域。这种推力推动了三个目标，依次解决心脏生理的更一般领域，每个领域都具有根本和治疗的影响。 （1）通过CA2+/CAM阐明心脏L型CA通道的促进。与CDI相反，尽管Ca2+（CDF）开放的促进通道开放的独特过程仍然神秘，尽管它在以更快的心率增强心跳中可能作用。 CDF在心脏中的实际强度仍然不清楚，以及CDF是否具有模型实验系统中发现的丰富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的不完全分离严重使心脏的研究变得复杂。因此，我们将在心肌细胞中表达工程设计的L型通道（缺少CDI和二氢吡啶块）。在天然通道的二氢吡啶块中，重组通道的选择性分辨率将允许在天然环境中明确评估和机械解剖。 （2）定义心脏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不仅调节其结合的通道，而且这种凸轮还可以桥接L型通道的优先信号传导到CREB。在这里，我们将使用独特的方法（例如CAM/L型通道融合）来定义肌细胞中CREB信号传导的基本方面，以测试调节通道的cam是否对于触发CREB至关重要。基于光FRET的CREB激活传感器还有望快速的CA2+进入模式和CREB激活的时间相关性。 （3）估计心脏细胞中L型通道附近局部内源凸轮的浓度。由于对局部Ca2+通过L型通道响应的CAM可能是最终触发CREB和其他核因子的启动CA2+传感器，因此从通道中限制局部Ca2+信号的CAM数量是下游信号传导强度的关键。在这里，我们将利用与聚合物链理论的CAM/L型通道融合，以估计内源性CAM近通道的局部浓度。初步结果暗示了MM浓度，这表明当地凸轮的“学校”位于渠道附近。总体而言，该建议将回答心脏中CAM/CA渠道生理的基本未知数。