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

• 批准号: 8793797
• 负责人: WILLIAM A CATTERALL
• 金额: $ 38.05万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-15 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8793797
• 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; Health; 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; receptor; reconstitution; research study; response; ventricular hypertrophy; voltage

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）通道启动心肌细胞的兴奋-收缩偶联。交感神经系统的刺激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）。PKA磷酸化Cav1.2通道并增加其活性，这有助于增加跳动率和收缩力，以响应运动，压力和恐惧。Cav1.2通道活性也受电压依赖性增强和Ca依赖性易化的调节，PKA和Ca/钙调蛋白依赖性蛋白激酶II（CaMKII）的磷酸化参与了这一调节。我们最近的研究揭示了PKA对Cav1.2通道调控的意想不到的复杂性。首先，在急性分离的心室肌细胞中，PKA锚定到远端C-末端结构域（DCT）需要A激酶锚定蛋白（AKAP）。第二，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通道分离。第三，蛋白水解处理的DCT非共价结合到近端C-末端结构域，并抑制Cav1.2通道活性。这种具有非共价结合的DCT、AKAP和PKA的自身抑制性信号传导复合物是调节PKA的主要底物，PKA使靠近这两半C末端相互作用位点的通道磷酸化并解除通道活性。我们已经使用蛋白质组学的方法来确定新的丝氨酸/苏氨酸残基，在体内磷酸化的反应？-肾上腺素能受体/PKA信号。酪蛋白激酶II对Thr 1704的磷酸化对于设置基础Cav1.2通道活性是重要的，而Ser 1700是PKA调节所必需的。小鼠中这些磷酸化位点的突变阻止了心室肌细胞中Cav1.2通道的肾上腺素能调节。此外，DCT缺失的小鼠具有显著的肥大和心力衰竭，表明这种自身抑制信号复合物是体内正常心血管功能所需的。我们提出的实验将达到三个目标。1.我们将使用无偏的免疫共沉淀和蛋白质组学方法来鉴定与心脏中Cav1.2结合的AKAP，并确定这些AKAP在通道调节中的功能作用。2.我们将分析电压依赖性增强和CaMK II依赖性促进全长，截断，截断+DCT通道在转染细胞中使用突变体在Ser 1700网站，以确定其功能作用，我们将定义该网站在体内使用S1700 A小鼠的功能作用。3.我们将研究全长，截短和截短+DCT Cav1.2通道水平的变化及其与AKAP在心功能衰竭的肾上腺素能过度刺激模型中的相互作用，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S1700 A小鼠来定义Ser 1700磷酸化在体内肥大和心力衰竭中的作用。这些研究将增加对交感神经系统调节心脏的理解，并对心力衰竭中Cav1.2信号复合物的分子和功能变化提供重要的新见解。