Calmodulin/Ca channel physiology in heart

总的推力是部署新的分子结构-许多灵感来自通道机制的研究-为发现基本的，新的访问领域的钙调素/钙通道生理在心脏。这一推动力推动了三个目标，相继解决了心脏生理学的更普遍的领域，每个领域都具有基础和治疗意义。(1)阐明Ca 2 +/CaM对心肌L型钙通道的易化作用。与CDI相比，Ca 2+（CDF）促进通道开放的独特过程仍然是神秘的，尽管它可能在更快的心率下加强心跳。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的不完全分离严重地使心脏中的研究复杂化。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）。在天然通道的二氢吡啶阻断期间，重组通道的选择性解析将允许在天然环境中明确评估和机械解剖CDF。(2)确定心脏L型钙通道激活核CREB的能力。这样的Ca 2+信号似乎对心脏基因的动态调节至关重要。在神经元中，CaM不仅调节其所结合的通道，这种CaM还可以将L型通道的优先信号传导桥接至CREB。在这里，我们将定义CREB信号在肌细胞的基本方面，使用独特的方法，如钙调素/L型通道融合，以测试是否非常钙调素，调节通道是必不可少的触发CREB。CREB激活的基于FRET的光学传感器也保证了Ca 2+进入模式和CREB激活的快速时间相关性。(3)目的：测定心肌细胞L型通道附近局部内源性钙调素（CaM）的浓度。由于CaM对通过L型通道的局部Ca 2+内流的响应可能是最终触发CREB和其他核因子的起始Ca 2+传感器，因此参与来自通道的局部Ca 2+信号的CaM的数量是下游信号强度的关键。在这里，我们将利用钙调素/L型通道融合，与聚合物链理论，估计本地浓度的内源性钙调素通道附近。初步结果暗示在mM浓度，这表明一个“学校”的本地钙调素居住在附近的渠道。总的来说，这个提议将回答心脏中CaM/Ca通道生理学的基本未知数。

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.