Structural and Functional Studies of Potassium Channels by Solid State NMR

通过固态核磁共振研究钾通道的结构和功能

基本信息

  • 批准号:
    10659941
  • 负责人:
  • 金额:
    $ 47.18万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2009
  • 资助国家:
    美国
  • 起止时间:
    2009-09-30 至 2027-04-30
  • 项目状态:
    未结题

项目摘要

Potassium channels control membrane potential and signaling processes for humans and pathogens. Essentially all characterized K+ channels inactivate after opening due to a transmembrane allosteric process that appears to be a slow result of activation and involves residues near the selectivity filter in the pore domain (c-type inactivation). Since the activated state is the only conductive species, and is metastable, inactivation controls mean open time and thereby modulates function for many important channels and drug targets. For example, neurons use K+ channel inactivation kinetics to modulate their firing frequency, and inactivation kinetics in the channels of the human heart have strong effects on heart timing. Our research provided evidence in the K+ channel KcsA that the molecular basis of c-type inactivation is transmembrane allosteric coupling between the activation gate (H+ binding to the intracellular pH sensor) and the inactivation gate (K+ release from the extracellular selectivity filter). In recent efforts, we have delineated the mechanism for transmembrane allosteric control of channel activity by identifying residues that serve important roles in the allosteric response using NMR chemical shifts and mutation. We showed that activation and opening directly lead to K+ loss in the selectivity filter for wild type, but not for inactivation-reduced mutants, for which thermodynamic coupling between opening and K+ affinity is reduced. Our studies use solid state NMR measurements on full-length wild-type channels in hydrated proteoliposomes and offer atomistic access to structure, as well as the dynamics and thermodynamics of ligand binding. Thus, studies in the last period offer support for the hypothesis that allosteric coupling between activation and inactivation is the basis for inactivation and channel timing. The studies also give support for the specific identities of the “hotspots”. (Aim 1) In the upcoming period, a definitive test will be based on mutants that are accelerated in inactivation, in the sense that these mutants increase the “timing” function rather than abolish it. Many of these faster-inactivating mutants are also of interest because of similarities or analogies to eukaryotic channels that are fast-inactivating. (Aim 2) We plan to probe the conformational dynamics of the activated open state of the channel with recently developed NMR methods, to identify spontaneous conversion to early intermediates of inactivation. Specific methods developed in the last period allow us to carry out studies of the dynamics of key carbonyl and aromatic groups. Recent breakthroughs in the sensitivity of solid state NMR methods will be harnessed so that controls can be performed to test hypothesized relationships between dynamics and function, for example from molecular dynamics simulations. (Aim 3) Finally, in Ktr, a related channel that has been identified as a drug target for many pathogenic bacteria, we plan to clarify ligand-channel interactions determining the binding location of promising lead compounds. We will also characterize the inactivation mechanism and determine the consequence to allostery, inactivation and function when ligands bind. A clearer understanding of the thermodynamics and dynamics in these exemplars of transmembrane allostery is likely to lead to clarification of broad biophysical principles, as well as specific insights into small molecule modulators of disease-related channel function.
钾通道控制膜电位和人类和病原体的信号传导过程。基本上所有 特征性K+通道开放后,由于跨膜变构过程,这似乎是一个 活化的缓慢结果,并涉及孔域中选择性过滤器附近的残基(c型失活)。以来 活化状态是唯一的导电物质,并且是亚稳态的,失活控制平均开放时间, 从而调节功能 许多重要的通道和药物靶点。例如,神经元使用K+通道 调节其放电频率的失活动力学,以及人心脏通道中的失活动力学 对心脏计时有很大影响。我们的研究为K+通道KcsA提供了证据,表明 c型失活是激活门(H+结合到细胞内pH)之间的跨膜变构偶联 传感器)和失活门(K+从细胞外选择性过滤器释放)。在最近的努力中,我们已经划定了 通过识别起重要作用的残基来跨膜变构控制通道活性的机制 用核磁共振化学位移和突变来研究变构反应。我们发现激活和开放直接导致 K+损失的选择性过滤野生型,但不是失活减少突变体,热力学耦合 开放和K+亲和力之间的关系降低。我们的研究使用固态核磁共振测量全长野生型 通道,并提供原子访问的结构,以及动力学和 配体结合的热力学因此,上一时期的研究为变构偶联的假说提供了支持, 激活和失活之间的关系是失活和通道定时的基础。这些研究也支持了 “热点”的具体身份。(Aim 1)在即将到来的时期,一个明确的测试将基于突变体, 在失活中加速,在这个意义上,这些突变体增加了“定时”功能,而不是消除它。 这些更快失活的突变体也是令人感兴趣的,因为它们与真核细胞通道相似或类似, 快速灭活。(Aim 2)我们计划探测通道激活开放状态的构象动力学, 最近开发的NMR方法,以确定自发转化为早期中间体的失活。具体 在上一阶段开发的方法使我们能够对关键羰基和芳香基团的动力学进行研究。 最近在固态核磁共振方法的灵敏度方面的突破将被利用,以便进行控制。 测试动力学和功能之间的假设关系,例如来自分子动力学模拟。 (Aim 3)最后,在Ktr(已被确定为许多致病菌的药物靶标的相关通道)中,我们计划 阐明配体-通道相互作用决定有前途的先导化合物的结合位置。我们还将 表征失活机制,并确定变构、失活和功能的后果, 配体结合。更清楚地了解这些跨膜范例的热力学和动力学 变构可能导致对广泛的生物物理学原理的澄清,以及对小分子的具体见解 疾病相关通道功能的调节剂。

项目成果

期刊论文数量(5)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Investigation of slow molecular dynamics using R-CODEX.
  • DOI:
    10.1016/j.jmr.2012.05.019
  • 发表时间:
    2012-09
  • 期刊:
  • 影响因子:
    2.2
  • 作者:
    Li, Wenbo;McDermott, Ann
  • 通讯作者:
    McDermott, Ann
Contribution of protein conformational heterogeneity to NMR lineshapes at cryogenic temperatures.
低温下蛋白质构象异质性对 NMR 线形的贡献。
Protein linewidth and solvent dynamics in frozen solution NMR.
  • DOI:
    10.1371/journal.pone.0047242
  • 发表时间:
    2012
  • 期刊:
  • 影响因子:
    3.7
  • 作者:
    Siemer AB;Huang KY;McDermott AE
  • 通讯作者:
    McDermott AE
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ANN E MCDERMOTT其他文献

ANN E MCDERMOTT的其他文献

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{{ truncateString('ANN E MCDERMOTT', 18)}}的其他基金

HIGH FIELD/HIGH FREQUENCY ESR FOR STUDYING DNP IN BIOMEMBRANES
用于研究生物膜中 DNP 的高场/高频 ESR
  • 批准号:
    8364114
  • 财政年份:
    2011
  • 资助金额:
    $ 47.18万
  • 项目类别:
DYNAMIC NUCLEAR POLARIZATION SOLID STATE NMR SPECTROMETER FOR BIOMOLECULAR STUDIE
用于生物分子研究的动态核偏振固态核磁共振波谱仪
  • 批准号:
    7839443
  • 财政年份:
    2010
  • 资助金额:
    $ 47.18万
  • 项目类别:
Structural and Functional Studies of Potassium Channels by Solid State NMR
通过固态核磁共振研究钾通道的结构和功能
  • 批准号:
    10460945
  • 财政年份:
    2009
  • 资助金额:
    $ 47.18万
  • 项目类别:
Structural and Functional Studies of Channels and Pumps by Solid State NMR
通过固态核磁共振研究通道和泵的结构和功能
  • 批准号:
    8325732
  • 财政年份:
    2009
  • 资助金额:
    $ 47.18万
  • 项目类别:
Structural and Functional Studies of Potassium Channels by Solid State NMR
通过固态核磁共振研究钾通道的结构和功能
  • 批准号:
    8760232
  • 财政年份:
    2009
  • 资助金额:
    $ 47.18万
  • 项目类别:
Structural and Functional Studies of Channels and Pumps by Solid State NMR
通过固态核磁共振研究通道和泵的结构和功能
  • 批准号:
    7941916
  • 财政年份:
    2009
  • 资助金额:
    $ 47.18万
  • 项目类别:
Structural and Functional Studies of Channels and Pumps by Solid State NMR
通过固态核磁共振研究通道和泵的结构和功能
  • 批准号:
    8142738
  • 财政年份:
    2009
  • 资助金额:
    $ 47.18万
  • 项目类别:
Structural and Functional Studies of Potassium Channels by Solid State NMR
通过固态核磁共振研究钾通道的结构和功能
  • 批准号:
    9117619
  • 财政年份:
    2009
  • 资助金额:
    $ 47.18万
  • 项目类别:
Structural and Functional Studies of Potassium Channels by Solid State NMR
通过固态核磁共振研究钾通道的结构和功能
  • 批准号:
    10021668
  • 财政年份:
    2009
  • 资助金额:
    $ 47.18万
  • 项目类别:
Structural and Functional Studies of Potassium Channels by Solid State NMR
通过固态核磁共振研究钾通道的结构和功能
  • 批准号:
    10224775
  • 财政年份:
    2009
  • 资助金额:
    $ 47.18万
  • 项目类别:

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