Ion channel regulation by macromolecular complexes

描述(申请人提供)：蛋白质磷酸化调节许多 细胞功能包括离子通道功能。在最大的离子中 通道是钙释放通道/兰尼定受体，它在心脏 和骨骼肌是兴奋收缩偶联所必需的。 蛋白激酶对心脏兰尼定受体(RyR2)的过度磷酸化作用 A(PKA)在人类心力衰竭中导致通道功能缺陷。 由于RyR过度磷酸化而引起的心脏钙处理的改变可能 部分原因是收缩和舒张期功能异常 心律失常。RyR2大分子复合体的形成涉及到 适配蛋白/激酶和磷酸酶在离子通道上的募集。 这些信号分子的局部靶向是通过特定的 连接蛋白通过亮氨酸/异亮氨酸拉链与通道结合 域名。我们最近发现RyR2与RyR2形成大分子络合物 MAKAP-PKA和两种磷酸酶(PP1通过接头蛋白、刺激性蛋白 和PP2A通过未知的接头蛋白)。这个项目的目标是 是为了研究接头蛋白招募的基础机制 和激酶/磷酸酶对心脏离子通道的作用。因为有几个心脏离子 通道(RyR2，L型钙通道A1C亚基，KvLQT1)有亮氨酸拉链 基序和被磷酸化调节，理解机制(S) 通过亮氨酸拉链靶向特定的调节剂可能会导致一种新的治疗模式 离子通道调节的研究。提出了三个具体目标来 阐明RyR2相关磷酸酶的靶向和功能作用： (1)PP1/刺亲素相互作用机制的表征 RyR2；(2)亮氨酸拉链基序的鉴定 负责PP2A/RyR2相互作用的靶向接头蛋白；以及(3) 磷酸酶锚定作用的功能表征 RyR2使用特定干扰本地信令的结构的表达。 这些发现可能会导致新的和特定的治疗方法来预防心脏 心力衰竭和心律失常的发生。我们计划利用定点突变来 进一步了解接头蛋白/磷酸酶靶向RyR2和 以确定磷酸酶的关联性和功能是否 在疾病状态下受到监管。我们将基于我们的 发现这些调节蛋白的结合部位，以测试 将PP1和/或PP2A靶向RyR负责 该通道的去磷酸化。我们预期拟议的研究将会 细胞内钙释放通道，将提供新的信息， 可能导致开发新的药理学方法来治疗 治疗心力衰竭和心律失常，可作为治疗心力衰竭和心律失常的模型 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.