Molecular choreography of CaV1.2 channels in the aging myocardium

项目摘要 在这个应用程序中概述的实验提出了一个新的范例，其中我们添加了一个新的复杂层 目前对β-肾上腺素能受体（βAR）介导的CaV1.2通道和EC-1的调节的理解， 偶合器.我们认为β AR的刺激启动了CaV1.2通道丰度的动态增加， 增强CaV1.2通道的合作门控，以及年轻人肌膜中的从头耦合子形成 心室肌细胞，以放大Ca 2+流入这些细胞，并在高需求时调节EC偶联。我们 初步数据表明，一个预先合成的肌膜下，CaV1.2通道， 囊泡/内体，存在于心肌细胞中，并且可以在高血压时被动员到肌膜。 代谢或血液动力学需求。我们假设这些“新”通道插入肌膜 以PKA和CaMK II介导的磷酸化依赖方式发生，而随后的内化 当需求减少时，移动的池的减少以CaN介导的去磷酸化依赖性方式发生。 进一步的初步数据表明，含有BAR结构域的蛋白质BIN 1，编排这种反应， 在微管和肌动蛋白介导的CaV1.2靶向肌膜和运输通道中发挥作用 并将其回收到细胞表面。引人注目的是，这一动态监管过程 在从老年小鼠分离的心室肌细胞中，我们发现心脏BIN 1蛋白水平几乎 翻了一倍随着年龄的增长，大脑中BIN 1的表达增加与阿尔茨海默病和缺陷有关 称为“内体运输堵塞”的细胞内运输。我们假设，BIN 1水平的增加与 衰老，可能导致心室肌细胞内类似的内体交通堵塞，导致基础CaV1.2增加 在肌膜上的表达和容易插入的通道池的耗尽。我们提出了一个模型， BIN 1在心室肌细胞中作为CaV1.2通道传递到肌膜的枢纽，并提示 随着年龄的增长，CaV1.2通道的分布和活性发生改变，对βAR刺激的反应性降低 是由BIN 1表达的变化介导的。特异性目的1检验了βAR激活刺激 肌膜CaV1.2通道丰度和聚集的年龄依赖性动态增加。具体目标 2检验了BIN 1表达的变化是CaV1.2动力学改变和保留的基础的假设。 渠道老化。最后，具体目标3检验了CaV1.2通道中年龄相关差异的假设， 心室肌细胞的动力学和运输导致“战斗或逃跑”期间EC偶联受损 反应为了实现这些目标，我们采用了多方面的方法，使用最先进的方法， 分析包括超分辨率成像、膜片钳电生理学、TIRF和共聚焦成像

Project Summary Experiments outlined in this application, suggest a novel paradigm, in which we add a new layer of complexity to the current understanding of β-adrenergic receptor (βAR)-mediated regulation of CaV1.2 channels and EC- coupling. We propose that stimulation of βARs initiates dynamic augmention of CaV1.2 channel abundance, enhanced cooperative gating of CaV1.2 channels, and de novo couplon formation in the sarcolemma of young ventricular myocytes, to amplify Ca2+ influx into these cells and tune EC-coupling in times of high demand. Our preliminary data suggest that a pre-synthesized pool of sub-sarcolemmal, CaV1.2 channels-containing vesicles/endosomes, resides in cardiomyocytes and can be mobilized to the sarcolemma in times of high metabolic or hemodynamic demand. We hypothesize that insertion of these ‘new’ channels into the sarcolemma occurs in a PKA- and CaMKII-mediated phosphyorylation dependent fashion, while subsequent internalization of the mobile pool when demand decreases, occurs in a CaN-mediated dephosphorylation dependent manner. Further preliminary data suggests that the BAR-domain containing protein BIN1, choreographs this response, with roles in microtubule and actin mediated CaV1.2 targeting to the sarcolemma and in trafficking channels out of early endosomes and recycling them back to the cell surface. Strikingly, this dynamic regulatory process is absent in ventricular myocytes isolated from aged mice where we find cardiac BIN1 protein levels are almost doubled. Increased expression of BIN1 in the brain with aging is associated with Alzheimer’s Disease and defects in intracellular trafficking termed ‘endosomal traffic jams’. We hypothesize that increased levels of BIN1 with aging, could cause analogous endosomal traffic jams in ventricular myocytes, leading to enhanced basal CaV1.2 expression at the sarcolemma and depletion of the readily insertable pool of channels. We propose a model in which BIN1 acts as a hub for CaV1.2 channel delivery to the sarcolemma in ventricular myocytes, and suggest that altered distribution and activity of CaV1.2 channels, and reduced responsivity to βAR stimulation with aging is mediated by changes in BIN1 expression. Specific Aim 1 tests the hypothesis that βAR activation stimulates age-dependent dynamic augmentation of sarcolemmal CaV1.2 channel abundance and clustering. Specific Aim 2 tests the hypothesis that changes in BIN1 expression underlie the altered dynamics and retention of CaV1.2 channels with aging. Finally, specific Aim 3 tests the hypothesis that age-related differences in CaV1.2 channel dynamics and trafficking in ventricular myocytes leads to impaired EC-coupling during the ‘fight or flight’ response. To achieve these aims, we employ a multi-faceted approach using state-of-the-art methods and analyses including super-resolution imaging, patch clamp electrophysiology, TIRF and confocal imaging