Calmodulin/Ca Channel Physiology in Heart

总的推动力是部署新的分子结构-许多灵感来自通道机制研究- 发现了心脏中钙调素/钙通道生理学的基本的、新的可及领域。这种推力 三个目标，依次解决心脏生理学的更普遍的领域，每一个都有基本的， 治疗意义(1)阐明Ca 2 +/CaM对心肌L型钙通道的易化作用。相比之下， CDI是钙离子易化通道开放的一个独特过程，尽管其可能的作用仍是一个谜 在加速心跳的过程中增强心跳。目前尚不清楚民防部队在心脏的实际力量，以及是否 CDF共享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 工程化的重组L型通道缺乏CDI，从而允许CDF的最大分辨率。通过 相反，CDF与CDI的不完全分离严重地使心脏中的研究复杂化。因此，我们将表达 工程化的L型通道（缺乏CDI和二氢吡啶阻断）。二氢吡啶阻断期间 重组通道的选择性分辨率将允许明确的评估， 在自然环境中对CDF进行机械性剥离。(2)为了定义心脏L型钙通道的能力， 激活核CREB。这种Ca 2+信号对心脏基因的动态调节至关重要.在 在神经元中，CaM不仅调节它所结合的通道，这种CaM还可以桥接优先的 L型通道向CREB发信号。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 通道是触发CREB的关键。基于FRET的CREB激活光学传感器也有望快速 Ca 2+内流模式与CREB激活时间相关性(3)估计当地的浓度 心脏细胞L型通道附近的内源性钙调素。由于钙调素对局部Ca 2+内流的反应， 通道可能是最终触发CREB和其他核因子的起始Ca 2+传感器， 钙调素参与局部钙信号的传递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，与聚合物链理论，以估计内源性CaM的局部浓度 靠近渠道。初步结果暗示mM浓度，表明当地CaMs的“学校”位于附近。 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 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.