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

描述（由申请人提供）：Ca 2+激活的钾通道，如小电导和中电导K+通道（SK和IK），在可兴奋组织中广泛表达。它们在调节Ca 2+对膜的兴奋性中起关键作用。与电压门控K+通道不同，SK/IK通道的激活仅由Ca 2+实现。钙调素（CaM），拴在通道的C-末端，作为高亲和力的Ca 2+传感器。CaM N端和C端分别有两个EF-臂，是Ca 2+的高亲和力结合位点。Ca 2+介导的CaM和CaM结合结构域（CaMBD）之间的相互作用激活该通道。除了它们的生理作用之外，SK/IK通道还与临床异常有关。 因此，大量的工作致力于开发靶向SK/IK通道的小分子。 虽然这种2x2复合物的Ca 2+依赖性形成是Ca 2+依赖性激活的关键初始步骤， SK通道，几乎没有进展，如何结合的Ca 2+钙调素是耦合到SK通道的最终开放。SK通道受到第二信使的调节，最值得注意的是，蛋白激酶CK 2在T79处对CaM的磷酸化降低了通道激活的Ca 2+敏感性。到目前为止，仍然不清楚T79处的CaM磷酸化如何导致SK通道的抑制。磷酸肌醇（PI）在细胞信号传导中起主要作用。PI脂质，特别是PI（4，5）P2，可以通过以下途径调节通道活性： 它们与通道蛋白的直接相互作用，包括Kvs、Kir、KCNQ和Cav通道。然而，PI脂质是否/如何调节SK通道活性实际上是未知的。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，我们将解决以下问题：（1）对Ca与CaM结合的偶联和SK通道的机械打开的结构洞察。（二） 通过膜脂质PI（4，5）P2调节SK通道活性。(3)通过蛋白质磷酸化调节PIP 2对其的亲和力， 靶蛋白。(4)有或没有钙调素的整个SK通道的结构测定。从拟议的工作的结果将提供深入了解的分子机制的SK通道激活钙调素，PI脂质的通道门控的调节。 此外，我们的研究结果将表明，不同的信号级联的收敛使得在生理条件下通过PIP 2调节通道活性成为可能，通过降低PIP 2对磷酸化通道蛋白的亲和力。

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.