Structural insights into SK channel gating and its regulation by membrane lipids

描述(申请人提供)：钙激活的钾通道，如小电导和中导K+通道(SK和IK)，在可兴奋的组织中广泛表达。它们在钙离子对细胞膜兴奋性的调控中起着关键作用。与电压门控K+通道不同，SK/IK通道的激活完全由钙离子实现。钙调素(CaM)与钙通道C末端相连，是高亲和力的钙离子感受器。四个EF-Hand是高亲和力的钙结合部位，其中两个位于CaM的N-端(N-叶)，另外两个位于C-端(C-叶)。钙离子介导的CaM与CaM结合域(CaMBD)之间的相互作用激活了该通道。除了它们的生理作用外，SK/IK通道还与临床异常有关。因此，人们致力于开发以SK/IK通道为靶点的小分子。而这种2x2复合体的钙依赖形成是钙依赖激活的关键初始步骤 关于钙离子与CaM的结合如何与SK通道的最终开放相偶联的研究进展甚微。SK通道受第二信使的调节，最值得注意的是，蛋白激酶CK2使T79位CaM的磷酸化降低了钙离子对通道激活的敏感性。到目前为止，尚不清楚T79位CaM的磷酸化如何导致SK通道的抑制。磷脂酰肌醇(PI)在细胞信号转导中发挥重要作用。PI脂类，尤其是PI(4，5)P2，可通过 它们与通道蛋白直接相互作用，包括KVS、KIR、KCNQ和Cav通道。然而，PI脂类是否/如何调节SK通道的活动几乎是未知的。我们将使用结构生物学、计算生物学、分子生物学、生物物理学和电生理学的综合方法来解决这些问题，具体地说，我们将解决以下问题：(1)钙结合到CaM和SK通道机械开放的耦合的结构洞察。(2)膜脂对SK通道活性的调节，PI(4，5)P2。(3)通过蛋白质磷酸化调节PIP2对其亲和力的调节 靶蛋白。(4)带或不带凸轮的整个SK通道的结构确定。这项工作的结果将为深入了解CaM激活SK通道的分子机制，以及PI类脂对通道门控的调节提供依据。此外，我们的结果将表明，在生理条件下，通过降低PIP2与磷酸化通道蛋白的亲和力，不同信号级联的融合使得PIP2对通道活动的调节成为可能。

DESCRIPTION (provided by applicant): Ca2+-activated potassium channels, such as small- and intermediate-conductance K+ channels (SK and IK), are widely expressed in excitable tissues. They play pivotal roles in regulating membrane excitability by Ca2+. Unlike voltage-gated K+ channels, activation of SK/IK channels is achieved exclusively by Ca2+. Calmodulin (CaM), tethered to the channel C-terminus, serves as the high-affinity Ca2+ sensor. Four EF-hands, two located at the CaM N-terminus (N-lobe) and the other two at the C-terminus (C-lobe), are the high affinity Ca2+ binding sites. The Ca2+-mediated interaction between CaM and the CaM binding domain (CaMBD) activates the channel. In addition to their physiological roles, SK/IK channels have been implicated in clinical abnormalities. Consequently, a tremendous effort has been devoted to developing small molecules targeting SK/IK channels. While Ca2+-dependent formation of this 2x2 complex is a critical initial step for Ca2+-dependent activation of SK channels, little progress has been made on how binding of Ca2+ to CaM is coupled to eventual opening of the SK channel. SK channels are subjected to regulation by second messengers, most notably; phosphorylation of CaM at T79 by protein kinase CK2 reduces the Ca2+ sensitivity for channel activation. Until now, it remains unknown how phosphorylation of CaM at T79 results in inhibition of SK channels. Phosphoinositides (PIs) play a major role in cellular signaling. PI lipids, particularly PI(4,5)P2, can regulate the channel activities through their direct interactions with the channel proteins, including Kvs, Kir, KCNQ and Cav channels. However, it is virtually not known whether/how PI lipids may regulate SK channel activities We will use integrated approaches of structural biology, computational biology, molecular biology, biophysics and electrophysiology to address these issues, specifically, we will address the following questions: (1) Structural insight into the coupling of Ca binding to CaM and mechanical opening of SK channels. (2) Regulation of the SK channel activity by the membrane lipid, PI(4,5)P2. (3) Regulation, by protein phosphorylation, of the PIP2 affinity for its target proteins. (4) Structural determination of the entire SK channel with or without CaM. Results from the proposed work will provide insights into the molecular mechanisms underlying activation of SK channels by CaM, and regulation of the channel gating by PI lipids. Furthermore, our results will show that convergence of different signaling cascades makes regulation of channel activities by PIP2 possible under physiological conditions, by reducing the affinity of PIP2 for the phosphoryalted channel proteins.