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

SK 通道门控及其膜脂调节的结构见解

• 批准号: 8759975
• 负责人: JI-FANG ZHANG
• 金额: $ 31.03万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8759975
• 关键词: Address; Affinity; Binding; Binding Sites; Biochemical; Bioinformatics; Biological Models; Biology; Biophysics; Calmodulin; Calorimetry; Cell membrane; Cellular biology; Clinical; Complex; Computational Biology; Coupled; Coupling; Data; EF Hand Motifs; Electrophysiology (science); Elements; Excision; Ion Channel; Lipids; Lobe; Mechanics; Mediating; Membrane; Membrane Lipids; Membrane Proteins; Modeling; Molecular; Molecular Biology; Orthologous Gene; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphorylation; Physiological; Play; Potassium Channel; Process; Property; Proteins; Publications; Regulation; Roentgen Rays; Role; Second Messenger Systems; Signal Transduction; Structure; Testing; Tissues; Titrations; Voltage-Gated Potassium Channel; Work; X ray diffraction analysis; X-Ray Diffraction; base; biophysical techniques; casein kinase II; insight; molecular dynamics; novel; public health relevance; research study; second messenger; sensor; small molecule; structural biology

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.

描述（由申请人提供）：Ca2+活化的钾通道，例如小型和中间导电K+通道（SK和IK），在可兴奋的组织中广泛表达。他们在通过Ca2+调节膜兴奋性方面发挥关键作用。与电压门控的K+通道不同，SK/IK通道的激活仅由Ca2+实现。钙调蛋白（CAM）被束缚在C端通道C末端，用作高亲和力CA2+传感器。四个EF手，两个位于cam N端（N-LOBE），另外两个位于C端（C-Lobe），是高亲和力Ca2+结合位点。 Ca2+介导的CAM与CAM结合结构域（CAMBD）之间的相互作用激活了通道。除了它们的生理作用外，SK/IK通道还与临床异常有关。 因此，已经付出了巨大的努力，致力于开发针对SK/IK通道的小分子。 而这种2x2复合物的Ca2+依赖性形成是Ca2+依赖性激活的关键初始步骤 SK频道，关于CA2+与CAM的结合如何与最终开放SK通道的结合几乎没有取得进展。 SK渠道受第二使者的监管，最值得注意的是；蛋白激酶CK2在T79处CAM的磷酸化降低了CA2+敏感性以进行通道激活。到目前为止，在T79处的CAM磷酸化如何导致SK通道的抑制作用尚不清楚。磷酸肌醇（PIS）在细胞信号传导中起主要作用。 PI脂质，尤其是PI（4,5）P2，可以通过 它们与通道蛋白的直接相互作用，包括KVS，KIR，KCNQ和CAV通道。但是，几乎不知道PI脂质是否可以调节SK通道活动，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，具体来说，我们将解决以下问题：（1）（1）结构性见解与SK和机器机器机械开放的CA结合到CA的结合。 （2）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PIP2的亲和力 靶蛋白。 （4）具有或没有CAM的整个SK通道的结构测定。拟议工作的结果将提供有关CAM通过CAM激活SK通道的分子机制的见解，并通过PI脂质调节通道门控。 此外，我们的结果将表明，不同信号传导级联反应的收敛性通过降低PIP2对磷酸化通道蛋白的亲和力在生理条件下通过PIP2可能调节通道活动。