Regulation of Cardiac Calcium Channels by an Autoinhibitory Signalling Complex

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。刺激交感神经系统可激活肾上腺素能受体、腺苷环化酶和cAMP依赖的蛋白激酶(PKA)。PKA使Cav1.2通道磷酸化并增加其活性，这有助于增加运动、压力和恐惧的搏动频率和收缩力量。Cav1.2通道活性还受电压依赖性增强和钙依赖性易化的调节，而PKA和钙/钙调蛋白依赖性蛋白激酶II(CaMKII)的磷酸化参与了这一调节。我们最近的研究发现，PKA对Cav1.2通道的调控出人意料地复杂。首先，在急性分离的心室肌细胞中，需要一个A激酶锚定蛋白(AKAP)将PKA锚定到远端C-末端结构域(DCT)。第二，体内的蛋白质降解过程切断了靠近其中心的C-末端，潜在地将DCT与Cav1.2通道分开。第三，经过蛋白降解处理的DCT以非共价方式结合到近端的C-末端结构域，并抑制Cav1.2通道的活性。这种与DCT、AKAP和PKA非共价结合的自抑制信号复合体是调节PKA的主要底物，它使C末端这两个半部分相互作用的部位附近的通道磷酸化，并抑制通道的活性。我们已经使用蛋白质组学方法来鉴定新的丝氨酸/苏氨酸残基，这些残基在体内被磷酸化，以响应β-肾上腺素能受体/PKA信号。酪蛋白激酶II对Thr1704的磷酸化对于设定基础Cav1.2通道活性是重要的，而丝氨酸1700是被PKA调节所必需的。小鼠体内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缺失的小鼠有明显的肥大和心力衰竭，表明这种自我抑制信号复合体是体内正常心血管功能所必需的。我们提议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，并确定这些AKAP在通道调节中的功能。2.我们将利用Ser1700位点的突变来分析电压依赖的增强作用和CaMKII依赖的全长、截短和截短的+DCT通道的易化，以确定其功能作用，并将在体内使用S1700A小鼠确定该位点的功能作用。3.在心力衰竭的肾上腺素能过度刺激模型中，我们将检测全长、截短和截短+DCT Cav1.2通道水平的变化及其与AKAP的相互作用。在该模型中，我们的初步研究揭示了Cav2.1的实质性分子重构。我们将使用我们的S1700A小鼠来确定Ser1700的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对心脏调节的了解，并为心力衰竭中Cav1.2信号复合体的分子和功能变化提供必要的新见解。

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.