Role of spectrin/ankyrin-G complex in myocyte signaling and cardiac excitability

项目摘要 离子通道和转运蛋白控制带电离子穿过细胞膜的运动。在 心脏，这些蛋白质的协调活动调节跨膜电化学梯度， 控制去极化/复极化，从而控制心脏兴奋性。离子通道/转运蛋白的正常功能 需要定义的生物物理特性以及定义的精确表达，组织和调节。 膜结构域我们最近的发现支持了心律失常的新基础，而不是基于突变， 影响通道生物物理学，而是对正确表达所需的蛋白质的突变， 可兴奋膜上离子通道和转运蛋白的局部调节。 Ca2 +/钙调蛋白依赖性蛋白激酶II（CaMKII）是一种多功能信号分子， 在心脏生理学和心脏病中的重要作用。类似于蛋白激酶A（PKA）和蛋白激酶C (PKC)CaMKII调节整个细胞中靶底物宿主的磷酸化状态。 然而，与PKA和PKC依赖的局部信号传导形成鲜明对比的是，关于局部信号传导， CaMKII靶向肌细胞。事实上，虽然已经提出了少数CaMKII结合伴侣， 调节局部信号传导，没有CaMKII靶向蛋白已在动物模型中验证。 我们的初步数据支持一种新的CaMKII靶向心肌细胞的分子机制 闰盘bIV血影蛋白，一种肌动蛋白和锚定蛋白G相关分子，与CaMKIId相关， 体外和体内。bIV血影蛋白以前仅在神经系统中研究，现在在心脏中表达 与血管紧张素-G结合，并将CaMK II靶向肌细胞闰盘，以直接调节局部电压门控Na + 渠道活动。值得注意的是，我们已经确定了一个以前难以捉摸的CaMKII磷酸化位点的Nav1.5 （S571）调节通道活性。bIV血影蛋白CaMKII结合缺陷的靶向小鼠模型 基序缺乏闰盘CaMK II表达，导致Nav1.5 S571磷酸化减少，Nav1.5 S571磷酸化改变， 通道活性、动作电位异常和整个动物心血管表型。这些 初步数据强烈支持bIV血影蛋白是以前未被认识的体内CaMKII靶向蛋白， 以及我们的中心假设，即bIV血影蛋白为基础的闰盘复合物提供了关键的信号传导， 和正常心脏功能的结构作用。此外，这些数据有力地表明， 抑制bIV血影蛋白/CaMKII相互作用将作为一种新的，高度特异性的机制， 抑制心脏中的持续性INa以治疗心律失常。虽然这项建议将主要侧重于国际清算银行的作用， 血影蛋白靶向的CaMKII在调节Nav1.5中，我们假设bIV血影蛋白复合物将发挥更广泛的作用。 在肌细胞闰盘的局部信号传导和结构调节中的作用。我们预测这些发现 将为体内局部CaMKII靶向提供第一个数据，为CaMKII的作用提供第一个数据， Nav1.5 S571用于健康和疾病中的心脏INa调节，定义了血管紧张素-G和bIV血影蛋白在 心脏信号，膜结构和功能，并最终确定一种新的和潜在的治疗 抑制心脏中持续性INa以治疗心律失常的机制。

Project Summary Ion channels and transporters control the movement of charged ions across cell membranes. In the heart, the coordinate activities of these proteins regulate the transmembrane electrochemical gradient to control depolarization/repolarization, and thus cardiac excitability. Normal function of ion channels/transporters requires defined biophysical properties as well as precise expression, organization, and regulation in defined membrane domains. Our recent findings support a new basis for arrhythmia based not on mutations which affect channel biophysics, but instead on mutations to proteins which are required for proper expression and local regulation of ion channels and transporters at excitable membranes. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional signaling molecule with critical roles in cardiac physiology and heart disease. Similar to protein kinase A (PKA) and protein kinase C (PKC), CaMKII regulates the phosphorylation status of a host of target substrates throughout the cell. However, in striking contrast to PKA- and PKC-dependent local signaling, little is known regarding local CaMKII targeting in myocytes. In fact, while a handful of CaMKII-binding partners have been proposed to regulate local signaling, no CaMKII-targeting proteins have been validated in animal models. Our preliminary data support a novel molecular mechanism for CaMKII targeting to the myocyte intercalated disc. bIV spectrin, an actin- and ankyrin-G-associated molecule, associates with CaMKIId both in vitro and in vivo. bIV spectrin, previously only studied in the nervous system, is expressed in heart, associates with ankyrin-G, and targets CaMKII to the myocyte intercalated disc to directly regulate local voltage-gated Na+ channel activity. Notably, we have identified a previously elusive CaMKII phosphorylation site on Nav1.5 (S571) that regulates channel activity. A targeted mouse model deficient in the bIV spectrin CaMKII-binding motif lacks intercalated disc CaMKII expression resulting in reduced Nav1.5 S571 phosphorylation, altered Nav channel activity, action potential abnormalities, and whole animal cardiovascular phenotypes. These preliminary data strongly support bIV spectrin as a previously unrecognized in vivo CaMKII-targeting protein, as well as our central hypothesis that the bIV spectrin-based intercalated disc complex provides critical signaling and structural roles for normal cardiac function. Furthermore, these data strongly suggest that targeted inhibition of the bIV spectrin/CaMKII interaction would serve as a novel, and highly specific mechanism to suppress persistent INa in the heart to treat arrhythmia. While this proposal will primarily focus on the role of bIV spectrin-targeted CaMKII in regulating Nav1.5, we hypothesize that the bIV spectrin complex will play a broader role in local signaling and structural regulation at the myocyte intercalated disc. We predict that these findings will provide the first data for local CaMKII targeting in vivo, provide the first data for the role of CaMKII and Nav1.5 S571 for cardiac INa regulation in health and disease, define the role of ankyrin-G and bIV spectrin in cardiac signaling, membrane structure, and function, and finally define a novel and potentially therapeutic mechanism to suppress persistent INa in heart to treat arrhythmia.