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

项目摘要 离子通道和转运体控制带电离子在细胞膜上的移动。在 心脏，这些蛋白质的协调活动调节跨膜电化学梯度，以 控制去极化/复极化，从而控制心脏兴奋性。离子通道/转运体的正常功能 需要明确的生物物理属性以及明确的表达、组织和规则 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 影响通道生物物理学，而不是对蛋白质的突变，这是正常表达和 兴奋性膜上离子通道和转运体的局部调节。 Ca2/钙调蛋白依赖的蛋白激酶II(CaMKII)是一种多功能信号分子，具有 在心脏生理学和心脏病中的关键作用。与蛋白激酶A(PKA)和蛋白激酶C类似 (PKC)，CaMKII调节整个细胞内一系列靶底物的磷酸化状态。 然而，与依赖PKA和PKC的局部信号转导形成鲜明对比的是，对局部信号转导知之甚少 CaMKII在心肌细胞中的靶向性。事实上，虽然有几个具有CaMKII约束力的合作伙伴被提议 调节局部信号，没有CaMKII靶向蛋白在动物模型中得到验证。 我们的初步数据支持CaMKII靶向心肌细胞的一种新的分子机制 间盘。Biv Spectrin是一种与肌动蛋白和骨架蛋白G相关的分子，与CaMKIId在 体外和体内。Biv血影蛋白以前只在神经系统中研究，在心脏中表达。 Ankyrin-G，并靶向CaMKII到心肌细胞间盘直接调节局部电压门控钠 通道活动。值得注意的是，我们已经在NaV1.5上发现了一个以前难以捉摸的CaMKII磷酸化位点 (S571)，其调节通道活动。Biv血影蛋白CaMKII结合缺陷的靶向小鼠模型 Motif缺乏插入盘状CaMKII表达导致NaV1.5 S571磷酸化降低，NAV改变 通道活动、动作电位异常和整个动物的心血管表型。这些 初步数据有力地支持Biv Spectrin是一种以前在体内未被识别的CaMKII靶向蛋白，如 以及我们的中心假设，即基于Biv血影蛋白的间盘复合体提供关键信号 以及正常心脏功能的结构作用。此外，这些数据强烈表明，有针对性的 抑制Biv Spectrin/CaMKII相互作用将成为一种新的、高度特异的机制 抑制心脏中持续存在的INA，以治疗心律失常。虽然本提案将主要侧重于Biv的作用 光影蛋白靶向CaMKII在调节NaV1.5中，我们假设Biv光影蛋白复合体将发挥更广泛的作用 在肌细胞间盘的局部信号和结构调节中的作用。我们预测这些发现 将为CaMKII在体内的局部靶向提供第一批数据，为CaMKII的作用提供第一批数据 NAV1.5 S571在健康和疾病中的心脏Ina调节，定义ankyrin-G和Biv Spectrin在其中的作用 心脏信号、膜结构和功能，并最终定义一种新的和潜在的治疗 抑制心脏持续性INA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.