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

描述(由申请人提供)：离子通道和转运体控制带电离子在细胞膜上的移动。在心脏中，这些蛋白的协调活动调节跨膜电化学梯度，以控制去极化/复极化，从而控制心脏的兴奋性。离子通道/转运体的正常功能需要明确的生物物理性质，以及在明确的膜结构域上的精确表达、组织和调控。我们最近的发现支持心律失常的新基础，它不是基于影响通道生物物理学的突变，而是基于蛋白质的突变，这些突变是正确表达和局部调节可兴奋膜上的离子通道和转运蛋白所必需的。钙/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具有重要作用。与蛋白激酶A(PKA)和蛋白激酶C(PKC)类似，CaMKII调节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，对心肌细胞中的局部CaMKII靶向知之甚少。事实上，虽然已经提出了几个CaMKII结合伙伴来调节局部信号传递，但没有任何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靶向心肌细胞间盘的一种新的分子机制。Biv Spectrin是一种肌动蛋白和骨架蛋白-G相关的分子，在体外和体内都与CaMKIId相关。之前只在神经系统研究的Biv Spectrin在心脏中表达，与ankyrin-G结合，并将CaMKII靶向肌细胞间盘，直接调节局部电压门控的Na+通道活动。值得注意的是，我们已经在NaV1.5(S571)上发现了一个先前难以捉摸的CaMKII磷酸化位点，它调节着通道的活性。缺乏Biv Spectrin CaMKII结合基序的靶向小鼠模型缺乏插入盘CaMKII表达，导致NaV1.5 S571磷酸化降低，NAV通道活性改变，动作电位异常，以及整个动物的心血管表型。这些初步数据有力地支持了Biv Spectrin作为一种以前在体内未知的CaMKII靶向蛋白，以及我们的中心假设，即基于Biv Spectrin的间盘复合体为正常的心脏功能提供了关键的信号和结构作用。此外，这些数据强烈表明，靶向抑制Biv Spectrin/CaMKII相互作用将成为一种新的、高度特异的机制来抑制心脏中持续的INA以治疗心律失常。虽然这项建议将主要集中在Biv Spectrin靶向的CaMKII在调节NaV1.5中的作用，但我们假设Biv Spectrin复合体将在心肌细胞间盘的局部信号和结构调控中发挥更广泛的作用。我们预测这些发现将为CaMKII在体内的局部靶向提供第一个数据，为CaMKII和NaV1.5 S571在心脏Ina调节中的作用提供第一个数据，确定ankyrin-G和Biv Spectrin在心脏信号、膜结构和功能中的作用，最终确定一个新的潜在的治疗机制来抑制心脏中持续的Ina来治疗心律失常。 与公共健康相关：离子通道/转运体的正常功能需要明确的生物物理性质，以及在明确的膜域中的精确表达、组织和调控。CaMKII是一种多功能信号分子，在心脏生理学和心脏病中具有重要作用。我们的研究将为CaMKII在体内的局部靶向提供第一个数据，为CaMKII和NaV1.5 S571在心脏Ina调节中的作用提供第一个数据，确定ankyrin-G和Biv Spectrin在心脏信号、膜结构和功能中的作用，最终确定一种新的潜在的治疗机制来抑制心脏中持续的Ina来治疗心律失常。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Normal function of ion channels/transporters requires defined biophysical properties as well as precise expression, organization, and regulation in defined membrane domains. CaMKII is a multifunctional signaling molecule with critical roles in cardiac physiology and heart disease. Our studies 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.