Regulation of Calcium Channels by Calmodulin

钙调蛋白对钙通道的调节

• 批准号: 7738498
• 负责人: STEEN E PEDERSEN
• 金额: $ 38.38万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7738498
• 关键词: Affect; Binding; Binding Sites; C-terminal; Calcium; Calcium Binding; Calcium Channel; Calmodulin; Cardiac; Complex; Crystallography; Cysteine; Detection; Disease; EF Hand Motifs; Energy Transfer; Engineering; Feedback; Goals; Heart; Hyperkalemic periodic paralysis; Hypokalemic periodic paralysis; Label; Lanthanoid Series Elements; Lobe; Location; Long QT Syndrome; Malignant hyperpyrexia due to anesthesia; Measurement; Measures; Mediating; Modeling; Molecular; Molecular Conformation; Muscle; Muscle Contraction; Mutation; Myocardial Infarction; N-terminal; Peptide Fragments; Process; Proteins; Regulation; Relative (related person); Research; Research Personnel; Resolution; Rest; Roentgen Rays; Site; Skeletal Muscle; Structure; Tail; Testing; Therapeutic; Timothy syndrome; Ursidae Family; base; human disease; luminescence resonance energy transfer; prevent; programs; research study; skeletal; tool; voltage

DESCRIPTION (provided by applicant): Voltage-gated calcium channels of the heart and of skeletal muscle are highly regulated to tune the function of each type of muscle. An integral part of the regulation is feedback inhibition and feedback activation of these channels by calcium entry through the channel itself. These feedback mechanisms, calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF), both depend on calcium binding to calmodulin and a change in conformation leading to a shift in the calmodulin binding site within the channel. It is still unknown how these two mechanisms can be regulated through the same protein at a complex binding site of the C-terminal tail of the channel. The hypothesis of this proposal is that feedback regulation occurs by shifting the conformation and binding of calmodulin within the C-terminal domain of the Ca2+-channel alpha-subunit. We will test this hypothesis through three specific aims: Aim 1 will examine the conformation of calmodulin in its apo-form, with no calcium occupancy, using high-precision, lanthanide-based energy transfer distance measurements between the lobes of calmodulin. The lobes will be localized by distance measurements to specific recognition sequences on the channel. In addition, X-ray crystallographic studies will determine the atomic-resolution structure of apo-calmodulin complexed with the recognition sequences. Aim 2 will utilize similar approaches to determine the binding sites for each calmodulin lobe and its conformation during partial and complete Ca2+ occupancy that occur during CDI. Aim 3 will determine conformation and sites of calmodulin binding in the presence of mutations known to affect CDI and CDF, using similar approaches. The results of these experiments will reveal the exact changes in conformation and location of calmodulin that occur during CDI and CDF and to provide a critical understanding of how these regulatory functions are initiated. The proteins under study in this proposal regulate the heart beat and skeletal muscle contraction. These channels are critically important in human disease; in the heart they are the targets of calcium blockers, which are routinely used to prevent heart attack. The proposed research provides information on how this channel is regulated, and, therefore, an opportunity to provide better therapeutics for diseases such as long QT syndrome, hyperkalemic periodic paralysis, malignant hyperthermia, and Timothy's syndrome.

描述(由申请人提供)：心脏和骨骼肌的电压门控钙通道受到高度调节，以调节每种肌肉的功能。调节的一个组成部分是反馈抑制和反馈激活这些通道，这些通道通过通道本身进入钙通道。这些反馈机制，即钙依赖失活(CDI)和钙依赖促进(CDF)，都依赖于钙与钙调蛋白的结合以及导致钙调素结合部位移动的构象变化。目前尚不清楚这两种机制如何通过通道C末端尾部的复杂结合位点上的同一蛋白质来调节。这一设想的假设是，反馈调节是通过改变钙通道α亚基C-末端结构域中钙调素的构象和结合而发生的。我们将通过三个具体的目标来验证这一假设：目标1将使用高精度的、基于稀土的钙调蛋白叶之间的能量转移距离测量来检查钙调蛋白在无钙占据的情况下以脱脂形式的构象。将通过对通道上的特定识别序列的距离测量来定位波瓣。此外，X射线结晶学研究将确定与识别序列络合的脱脂钙调蛋白的原子分辨结构。AIM 2将利用类似的方法来确定CDI期间发生的部分和完全钙占用过程中每个钙调蛋白叶的结合位置及其构象。目标3将使用类似的方法，在已知影响CDI和CDF的突变存在的情况下，确定钙调蛋白结合的构象和位置。这些实验的结果将揭示钙调素在CDI和CDF过程中的确切构象和位置的变化，并为了解这些调控功能是如何启动的提供关键的理解。这项研究中所研究的蛋白质调节心跳和骨骼肌收缩。这些通道在人类疾病中至关重要；在心脏中，它们是钙阻滞剂的靶标，钙阻滞剂通常用于预防心脏病发作。这项拟议的研究提供了有关该通道如何调节的信息，因此，有机会为长QT综合征、高钾性周期性麻痹、恶性高热和Timothy综合征等疾病提供更好的治疗方法。