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反馈抑制和反馈激活这些通道的钙通过通道本身进入。这些反馈机制，钙依赖性失活（CDI）和钙依赖性促进（CDF），都依赖于钙与钙调蛋白的结合，以及导致通道内钙调蛋白结合位点移位的构象变化。目前尚不清楚这两种机制是如何通过通道c端尾部复杂结合位点的同一蛋白来调节的。该建议的假设是，反馈调节是通过改变钙调蛋白在Ca2+通道α亚基的c端结构域的构象和结合而发生的。我们将通过三个具体目标来验证这一假设：目的1将使用高精度的、基于镧系元素的钙调蛋白叶之间的能量传递距离测量，在没有钙占用的情况下，研究钙调蛋白在载脂蛋白形式的构象。这些波瓣将通过距离测量定位到信道上的特定识别序列。此外，x射线晶体学研究将确定载脂蛋白钙调素配合识别序列的原子分辨率结构。Aim 2将利用类似的方法来确定每个钙调蛋白叶的结合位点及其在CDI期间发生的部分和完全Ca2+占用期间的构象。Aim 3将使用类似的方法，确定已知影响CDI和CDF的突变存在时钙调素结合的构象和位点。这些实验的结果将揭示CDI和CDF期间钙调蛋白构象和位置的确切变化，并提供这些调节功能如何启动的关键理解。这项研究中研究的蛋白质调节心脏跳动和骨骼肌收缩。这些渠道在人类疾病中至关重要；在心脏中，它们是钙受体阻滞剂的目标，钙受体阻滞剂通常用于预防心脏病发作。拟议的研究提供了该通道如何被调节的信息，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.