Regulation of Calcium Channels by Calmodulin

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通过钙通过通道本身进入这些通道的反馈抑制和反馈激活。这些反馈机制，钙依赖性失活（CDI）和钙依赖性易化（CDF），都依赖于钙结合钙调蛋白和构象的变化，导致在通道内的钙调蛋白结合位点的移位。目前还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处的相同蛋白质进行调节。该提议的假设是，反馈调节通过改变Ca 2+通道α亚基的C-末端结构域内的钙调蛋白的构象和结合而发生。我们将通过三个具体目标来测试这一假设：目标1将使用钙调素叶之间的高精度镧系元素能量转移距离测量来检查钙调素在其apo形式中的构象，没有钙占用。波瓣将通过距离测量定位到通道上的特定识别序列。此外，X射线晶体学研究将确定与识别序列复合的脱辅基钙调素的原子分辨率结构。目标2将利用类似的方法来确定结合位点的每个钙调素叶和它的构象在部分和完全的Ca 2+占用过程中发生的CDI。目标3将确定构象和钙调素结合位点的突变已知影响CDI和CDF的存在下，使用类似的方法。这些实验的结果将揭示在CDI和CDF过程中发生的钙调蛋白的构象和位置的确切变化，并提供这些调节功能是如何启动的关键理解。这项研究中的蛋白质调节心脏跳动和骨骼肌收缩。这些通道在人类疾病中至关重要;在心脏中，它们是钙阻滞剂的目标，钙阻滞剂通常用于预防心脏病发作。拟议的研究提供了关于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，恶性高热和蒂莫西综合征等疾病提供更好的治疗方法。

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.