Calmodulin/Ca channel physiology in heart

总的推动力是部署新的分子结构——许多来自通道机制研究的灵感——用于发现心脏中CaM/Ca通道生理学的基本的、新的可接近的领域。这一推动力驱动了三个目标，依次解决心脏生理学的更一般领域，每个领域都具有基础和治疗意义。(1)阐明Ca2+/CaM对心脏l型Ca通道的促进作用。与CDI相比，Ca2+ (CDF)促进通道打开的独特过程仍然是神秘的，尽管它可能在加快心率时加强心跳。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的不完全分离严重复杂化了心脏研究。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）。在二氢吡啶阻滞天然通道的过程中，重组通道的选择性分辨将允许在天然环境中对CDF进行明确的评估和机制解剖。(2)明确心脏l型Ca通道激活核CREB的能力。这种Ca2+信号似乎对心脏基因的动态调节至关重要。在神经元中，CaM不仅可以调节其结合的通道，还可以将l型通道的优先信号传导到CREB。在这里，我们将定义肌细胞中CREB信号传导的基本方面，使用独特的方法，如CaM/ l型通道融合，来测试调节通道的CaM是否对触发CREB至关重要。基于光学fret的CREB激活传感器也承诺Ca2+进入模式和CREB激活的快速时间相关性。(3)估计心脏细胞l型通道附近局部内源性CaM的浓度。由于通过l型通道响应局部Ca2+内流的cam可能是最终触发CREB和其他核因子的初始Ca2+传感器，因此从通道获取局部Ca2+信号的cam数量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，结合聚合物链理论，来估计通道附近内源性CaM的局部浓度。初步结果暗示了mM浓度，表明当地cam的“学校”位于通道附近。总的来说，这一建议将回答心脏中CaM/Ca通道生理学的基本未知。

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.