Calmodulin/Ca channel physiology in heart

总的主旨是部署新的分子结构--其中许多是从通道机制研究中获得的--以发现心脏中钙调素/钙通道生理学的基本的、新的可获得的领域。这一推力推动了三个目标，依次解决了心脏生理学的更一般领域，每个领域都具有基本和治疗意义。(1)阐明钙/钙调素对心肌L型钙通道的易化作用。与CDI相反，钙离子(CDF)促进通道开放的独特过程仍然是个谜，尽管它可能在以更快的心率加强心跳方面发挥作用。目前尚不清楚CDF在心脏中的实际强度，以及CDF是否具有在模型实验系统中发现的丰富的CaM信号特征。这些系统允许研究缺乏CDI的工程化重组L类型的通道，从而允许CDF的最大分辨率。相比之下，CDF和CDI的不完全分离使心脏研究严重复杂化。因此，我们将在心肌细胞中表达工程化的L类通道(缺乏CDI和二氢吡啶阻断)。在二氢吡啶阻断天然通道的过程中，对重组通道的选择性解析将允许在天然环境下对CDF进行明确的评估和机械解剖。(2)明确心肌L型钙通道激活核CREB的能力。这种钙信号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，还可能将L类通道的优先信号传递给CREB。在这里，我们将定义心肌细胞中CREB信号的基本方面，使用不同的方法，如CaM/L类型的通道融合来测试调节通道的CaM是否是触发CREB的关键。基于光学FRET的CREB激活传感器也可以实现钙离子进入模式和CREB激活的快速时间关联。(3)测定心肌细胞L型通道附近局部内源性CaM的浓度。由于通过L类通道对局部钙离子内流作出反应的CAM可能是最终触发CREB和其他核因子的起始钙感受器，因此从通道获得局部钙信号的CAM的数量是决定下游信号强度的关键。在这里，我们将利用CaM/L型通道融合，结合聚合链理论，估计通道附近内源CaM的局部浓度。初步结果暗示了mm的浓度，这表明有一群当地的凸轮驻扎在海峡附近。总体而言，这一建议将回答心脏钙调素/钙通道生理学的基本未知问题。

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.