Calmodulin/Ca Channel Physiology in Heart

总的主旨是部署新的分子结构-许多受到渠道机制研究的启发--用于 发现心脏钙调素/钙通道生理学的基本、新的可及领域。这个推力驱动 三个目标，依次解决更一般的心脏生理学领域，每个目标都有基本的和 治疗方面的影响。(1)阐明钙/钙调素对心肌L型钙通道的易化作用。与……形成对比 CDI是一种由钙离子(CDF)促进的通道开放的独特过程，尽管它可能起到作用，但仍是一个谜 以更快的心率加强心跳。目前尚不清楚CDF在内心的实际力量，以及 CDF共享在模型实验系统中发现的丰富的CaM信号功能。这些系统允许研究 缺乏CDI的工程化重组L类型通道，从而允许CDF的最大分辨率。通过 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，我们将表示 在心肌细胞中构建L型通道(缺乏胞二磷胆碱和二氢吡啶阻断)。二氢吡啶阻断期间 对于原生通道，重组通道的选择性解析将允许进行明确的评估和 本土化背景下对CDF的机械剖析。(2)明确心肌L型钙通道的功能。 启动核CREB。这种钙信号似乎对心脏基因的动态调节至关重要。在……里面 神经元，CaM不仅调节它所结合的通道，这样的CaM也可能桥接优先 L类通道对CREB的信令。在这里，我们将定义心肌细胞中CREB信号的基本方面，使用 独特的方法学，如CaM/L类型的通道融合，以测试是否正是调制 通道是触发CREB的关键。基于光学FRET的CREB激活传感器也有望快速 钙离子进入模式与CREB激活的时间相关性。(3)估算局部污染浓度 心肌细胞L型通道附近的内源性CaM。AS细胞对局部钙离子通过L型钙离子内流作出反应 通道可能是启动的钙感受器，最终触发CREB和其他核因子，数量 从通道获得局部钙信号的CAM是下行信号强度的关键。在这里，我们将利用 CaM/L型通道融合，用聚合链理论估计局部内源性CaM浓度 在海峡附近。初步结果暗示了毫米的浓度，这表明附近有一群当地的凸轮。 频道。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 Ca 2+ (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 Ca 2+ 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 Ca 2+ 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 Ca 2+ influx through L-type channels may be the initiatory Ca 2+ sensors that ultimately trigger CREB and other nuclear factors, the number of CaMs privy to the local Ca 2+ 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.