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（申请人提供）：Ca2+激活的钾通道，例如小电导和中电导K+通道（SK和IK），广泛表达于兴奋组织中。它们在调节 Ca2+ 膜兴奋性方面发挥着关键作用。与电压门控 K+ 通道不同，SK/IK 通道的激活完全由 Ca2+ 实现。钙调蛋白 (CaM) 与通道 C 末端相连，充当高亲和力 Ca2+ 传感器。四个 EF 手，两个位于 CaM N 末端（N 叶），另外两个位于 C 末端（C 叶），是高亲和力 Ca2+ 结合位点。 CaM 和 CaM 结合域 (CaMBD) 之间的 Ca2+ 介导的相互作用激活通道。除了其生理作用外，SK/IK 通道还与临床异常有关。 因此，人们付出了巨大的努力来开发针对 SK/IK 通道的小分子。 虽然这种 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 通道，在 Ca2+ 与 CaM 的结合如何与 SK 通道的最终打开相结合方面几乎没有取得任何进展。 SK 渠道受到第二信使的监管，最值得注意的是；蛋白激酶 CK2 在 T79 磷酸化 CaM 会降低 Ca2+ 通道激活的敏感性。迄今为止，T79 位点 CaM 磷酸化如何导致 SK 通道抑制仍不清楚。磷酸肌醇 (PI) 在细胞信号传导中发挥着重要作用。 PI 脂质，特别是 PI(4,5)P2，可以通过以下方式调节通道活性： 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，实际上尚不清楚PI脂质是否/如何调节SK通道活性。我们将利用结构生物学、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) 对 Ca 与 CaM 结合的耦合以及 SK 通道的机械打开的结构洞察。 (2)膜脂PI(4,5)P2对SK通道活性的调节。 (3) 通过蛋白质磷酸化调节 PIP2 对其的亲和力 目标蛋白质。 (4)有或没有CaM的整个SK通道的结构确定。这项工作的结果将深入了解 CaM 激活 SK 通道的分子机制，以及 PI 脂质调节通道门控的分子机制。 此外，我们的结果将表明，通过降低 PIP2 对磷酸化通道蛋白的亲和力，不同信号级联的汇聚使得 PIP2 在生理条件下调节通道活性成为可能。