Regulation of Cardiac Calcium Channels by an Autoinhibitory Signalling Complex

自抑制信号复合物对心脏钙通道的调节

基本信息

批准号：
8436650
负责人：
WILLIAM A CATTERALL
金额：
$ 38.63万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-05-15 至 2017-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8436650
关键词：
A kinase anchoring protein Action Potentials Address Adenylate Cyclase Adrenergic Agents Adrenergic Receptor Amino Acid Sequence Binding Binding Sites Biological Assay Brain C-terminal Calcium Calcium Channel Calcium Signaling Calcium/calmodulin-dependent protein kinase Cardiac Cardiac Myocytes Cardiovascular Physiology Cells Chemosensitization Co-Immunoprecipitations Complex Coupling Cyclic AMP-Dependent Protein Kinases Distal Down-Regulation Exercise Fright Heart Heart Contractilities Heart Rate Heart failure Human Hypertrophy Immunoblotting In Vitro Knock-in Mouse Knowledge Length Leucine Zippers Mass Spectrum Analysis Methods Modeling Molecular Mus Muscle Cells Mutant Strains Mice Mutate Mutation Oryctolagus cuniculus P-Q type voltage-dependent calcium channel Pathway interactions Phosphorylation Phosphorylation Site Positioning Attribute Process Protein Binding Protein Kinase Proteolytic Processing Proteomics Regulation Regulatory Pathway Relative (related person)Research Role Ryanodine Sarcoplasmic Reticulum Signal Pathway Signal Transduction Site Skeletal Muscle Specificity Stress Sympathetic Nervous System Terminator Codon Testing Up-Regulation Ventricular adrenergic calmodulin-dependent protein kinase II casein kinase II genetic association in vitro activity in vivo insight interest mouse model mutant novel prevent public health relevance receptor reconstitution research study response ventricular hypertrophy voltage

项目摘要

DESCRIPTION (provided by applicant): Voltage-gated calcium (Ca) channels initiate excitation-contraction coupling in cardiac myocytes. Stimulation of the sympathetic nervous system activates ¿-adrenergic receptors, adenylyl cyclase, and cAMP-dependent protein kinase (PKA). PKA phosphorylates Cav1.2 channels and increases their activity, which contributes to increased beating rate and contractile force in response to exercise, stress, and fear. Cav1.2 channel activity is also regulated by voltage-dependent potentiation and Ca-dependent facilitation, and phosphorylation by PKA and Ca/calmodulin-dependent protein kinase II (CaMKII) is involved in this regulation. Our recent research has revealed unexpected complexity in regulation of Cav1.2 channels by PKA. First, in acutely dissociated ventricular myocytes, an A Kinase Anchoring Protein (AKAP) is required for anchoring of PKA to the distal C- terminal domain (DCT). Second, in vivo proteolytic processing severs the C-terminus near its center, potentially separating the DCT from the Cav1.2 channel. Third, the proteolytically processed DCT binds noncovalently to the proximal C-terminal domain and inhibits Cav1.2 channel activity. This autoinhibitory signaling complex with noncovalently bound DCT, AKAP, and PKA is the primary substrate for regulation PKA, which phosphorylates the channel near the site of interaction of these two halves of the C-terminus and disinhibits channel activity. We have used proteomic methods to identify novel Ser/Thr residues that are phosphorylated in vivo in response to ¿-adrenergic receptor/PKA signaling. Phosphorylation of Thr1704 by casein kinase II is important for setting basal Cav1.2 channel activity, whereas Ser1700 is required for regulation by PKA. Mutation of these phosphorylation sites in mice prevents ¿-adrenergic regulation of Cav1.2 channels in ventricular myocytes. Moreover, mice in which the DCT is deleted have marked hypertrophy and heart failure, indicating that this autoinhibitory signaling complex is required for normal cardiovascular function in vivo. Our proposed experiments will address three aims. 1. We will use unbiased co-immunoprecipitation and proteomic methods to identify AKAPs that bind to Cav1.2 in the heart, and the functional role of these AKAPs in channel regulation will be determined. 2. We will analyze voltage-dependent potentiation and CaMKII-dependent facilitation of full-length, truncated, and truncated+DCT channels in transfected cells using mutants at the Ser1700 site to determine its functional role, and we will define the functional role of this site in vivo using S1700A mice. 3. We will examine changes in the levels of full-length, truncated, and truncated+DCT Cav1.2 channels and their interactions with AKAPs in the ¿-adrenergic hyperstimulation model of heart failure, in which our preliminary studies reveal substantial molecular remodeling of Cav2.1. We will use our S1700A mice to define the role of phosphorylation of Ser1700 in hypertrophy and heart failure in vivo. These studies will increase understanding of regulation of the heart by the sympathetic nervous system and give essential new insight into the molecular and functional changes in the Cav1.2 signaling complex in heart failure.

描述（由申请人提供）：电压门控钙（Ca）通道启动心肌细胞中的兴奋-收缩耦合，刺激交感神经系统激活。 -肾上腺素能受体、腺苷酸环化酶和 cAMP 依赖性蛋白激酶 (PKA) 磷酸化 Cav1.2 通道并增加其活性，这有助于增加对运动、压力和恐惧的反应的跳动率和收缩力。通道活性还受到电压依赖性增强和 Ca 依赖性促进以及 PKA 和 Ca/钙调蛋白依赖性蛋白激酶 II (CaMKII) 的磷酸化的调节我们最近的研究揭示了 PKA 调节 Cav1.2 通道的意外复杂性。首先，在急性分离的心室肌细胞中，需要 A 激酶锚定蛋白 (AKAP) 将 PKA 锚定到远端 C-。其次，体内蛋白水解加工切断其中心附近的 C 末端，可能将 DCT 与 Cav1.2 通道分开。第三，蛋白水解加工。 DCT 非共价结合近端 C 末端结构域并抑制 Cav1.2 通道活性。这种与非共价结合 DCT、AKAP 和 PKA 的自抑制信号复合物是调节 PKA 的主要底物，它使这些相互作用位点附近的通道磷酸化。我们使用蛋白质组学方法来鉴定体内磷酸化的新 Ser/Thr 残基。回应 ¿ - 酪蛋白激酶 II 的肾上腺素受体/PKA 信号传导对设置基础 Cav1.2 通道活性非常重要，而 PKA 的调节需要 Ser1700。小鼠中这些磷酸化位点的突变可防止 ¿ -心室肌细胞中Cav1.2通道的肾上腺素调节。此外，DCT被删除的小鼠具有明显的肥大和心力衰竭，这表明这种自抑制信号复合物是体内心血管正常功能所必需的。我们提出的实验将解决三个目标。 1. 我们将使用无偏免疫共沉淀和蛋白质组学方法来鉴定心脏中与 Cav1.2 结合的 AKAP，以及这些 AKAP 在通道调节中的功能作用。 2. 我们将使用 Ser1700 位点的突变体分析转染细胞中全长、截短和截短+DCT 通道的电压依赖性增强和 CaMKII 依赖性促进，以确定其功能作用，并定义其功能。使用S1700A小鼠体内该位点的作用3.我们将检查全长、截短和截短+DCT Cav1.2的水平的变化。渠道及其与 AKAP 的互动 ¿ -心力衰竭的肾上腺素过度刺激模型，其中我们的初步研究揭示了 Cav2.1 的实质性分子重塑。我们将使用我们的 S1700A 小鼠来定义 Ser1700 磷酸化在体内肥厚和心力衰竭中的作用。交感神经系统对心脏的调节，并为心力衰竭中 Cav1.2 信号复合物的分子和功能变化提供了重要的新见解。