Ion channel regulation by macromolecular complexes

描述（由申请人提供）：蛋白质磷酸化调节许多 包括离子通道功能的细胞功能。最大的离子 通道是钙释放通道/兰尼碱受体，在心脏中 和骨骼肌是兴奋收缩偶联所必需的。 蛋白激酶介导的心肌Ryanodine受体（RyR2）过度磷酸化 A（PKA）导致人类心力衰竭中通道的功能缺陷。 由于RyR的过度磷酸化导致的心脏钙处理的改变可能 部分原因是收缩和舒张功能异常， 胚胎发生RyR2大分子复合物的形成涉及 衔接蛋白/激酶和磷酸酶向离子通道的募集。 这些信号分子的局部靶向是通过特异性的 衔接蛋白通过亮氨酸/异亮氨酸拉链与通道结合 域.我们最近发现，RyR2形成大分子复合物， mAKAP-PKA和两种磷酸酶（PP1通过衔接蛋白， 和PP2A通过未知的衔接蛋白）。本项目的目标 是为了研究衔接蛋白的募集机制 和激酶/磷酸酶到心脏离子通道。自从几个心脏离子 通道（RyR2，L型Ca2+通道的a1c亚基，KvLQT 1）具有亮氨酸拉链 基序，并通过磷酸化调节，了解机制（S） 通过亮氨酸拉链靶向的特异性调节剂可能导致一种新的范例， 对离子通道调节的研究。提出了三个具体目标， 阐明RyR2相关磷酸酶的靶向和功能作用： (1)PP1/spinophilin相互作用机制的表征 （2）亮氨酸拉链基序的表征和RyR2的鉴定 负责PP2A/RyR2相互作用的靶向衔接蛋白;和（3） 磷酸酶锚定作用的功能表征 RyR2使用特异性破坏局部信号传导的构建体的表达。 这些发现可能会导致新的和具体的治疗，以防止心脏病 失败和失败。我们计划利用定点突变， 进一步了解衔接蛋白/磷酸酶靶向RyR2， 以确定磷酸酶的结合和功能是否 在疾病状态下进行调节。我们将利用基于我们的新战略， 这些调节蛋白的结合位点的发现，以测试是否 PP1和/或PP2A对RyR的靶向作用是导致 通道的去磷酸化。我们预计， 细胞内钙释放通道，将提供新的信息， 可能导致新的药理学方法的发展， 治疗心力衰竭和血管生成，并可作为模型， 其他心脏离子通道的研究。

DESCRIPTION (provided by applicant): Protein phosphorylation regulates numerous cellular functions including ion channel function. Among the largest ion channels are the calcium release channel/ryanodine receptors, which in cardiac and skeletal muscle are required for excitation contraction coupling. Hyperphosphorylation of the cardiac ryanodine receptor (RyR2) by protein kinase A (PKA) causes defective function of the channel in human heart failure. Altered calcium handling in the heart due to hyperphosphorylation of RyR may account for, in part, abnormal systolic and diastolic function and arrhythmogenesis. RyR2 macromolecular complex formation involves the recruitment of adaptor proteins/kinases and phosphatases to the ion channel. The local targeting of these signaling molecules is through the specific binding of adaptor proteins to the channel via leucine/isoleucine zipper domains. We have recently found that RyR2 forms a macromolecular complex with mAKAP-PKA and two phosphatases (PP1 through the adaptor protein, spinophilin and PP2A through an unknown adaptor protein). The objectives of this project are to examine the mechanisms that underlie the recruitment of adaptor proteins and kinases/phosphatases to cardiac ion channels. Since several cardiac ion channels (RyR2, a1c subunit of L-type Ca2+ channel, KvLQT1) have leucine zipper motifs and are regulated by phosphorylation, understanding the mechanism(s) of specific modulator targeting via leucine zippers may lead to a new paradigm for the study of ion channel regulation. Three specific aims are proposed to elucidate the targeting and functional role of RyR2-associated phosphatases: (1) Characterization of the mechanism by which PP1/spinophilin interacts with RyR2; (2) Characterization of the leucine zipper motif and identification of the targeting adaptor protein responsible for PP2A/RyR2 interaction; and (3) Functional characterization of the role of the anchoring of phosphatases to RyR2 using expression of constructs that specifically disrupt local signaling. These findings may lead to novel and specific therapies to prevent heart failure and arrhythmogenesis. We plan to utilize site-directed mutagenesis to further understand the targeting of adaptor proteins/phosphatases to RyR2 and to determine whether the association of and function of phosphatases are regulated in disease states. We will utilize novel strategies based upon our findings of the binding sites of these regulatory proteins to test whether targeting of PP1 and/or PP2A to RyR is (are) responsible for the dephosphorylation of the channel. We anticipate that the proposed studies of the intracellular calcium release channel, will provide new information that could lead to the development of novel pharmacological approaches to the treatment of heart failure and arrhythmogenesis and may serve as a model for the study of other cardiac ion channels.