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
• 负责人: ANN E MCDERMOTT
• 金额: $ 35.98万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10021668
• 关键词: Affect; Affinity; Amino Acids; Arrhythmia; Behavior; Binding; Cardiac; Cell Signaling Process; Chemicals; Communicable Diseases; Coupled; Coupling; Data; Disease; Distal; Drug Targeting; Event; Evolution; Exhibits; Freezing; Frequencies; Grant; Health; Heart; Human; Hydration status; Investigation; Ion Channel; Ions; Laboratories; Length; Life; Ligand Binding; Ligands; Long QT Syndrome; Malignant Neoplasms; Measurement; Mediating; Medical; Membrane; Membrane Potentials; Methods; Modeling; Molecular; Molecular Conformation; Mutagenesis; Mutation; Nature; Nervous System Physiology; Nervous system structure; Neurons; Participant; Pharmaceutical Preparations; Physiologic pulse; Physiological; Play; Potassium Channel; Process; Property; Protein Region; Regulation; Regulatory Element; Rest; Role; Signal Transduction; Site; Structure; Study models; Syndrome; System; Testing; Thermodynamics; Time; Work; base; design; experience; experimental study; extracellular; human pathogen; insight; mutant; nanosecond; potassium ion; receptor; response; sensor; solid state nuclear magnetic resonance; tool

Abstract Potassium channels control numerous signaling processes for humans and pathogens. Essentially all characterized K+ channels inactivate spontaneously after opening, due to a transmembrane allosteric process that acts to control mean open time. Inactivation modulates function for many important channels and drug targets: for example, neurons use K+ channel inactivation to modulate their firing frequency, and inactivation in the channels of the human heart has strong effects on heart timing. Our recent work provided evidence that the molecular basis of C type inactivation in KcsA is transmembrane allosteric coupling, where opening of the intracellular activation gate causes the extracellular selectivity filter to lose its affinity for K+. We showed that this transmembrane allosteric coupling is strong in the wild type channel in bilayers and absent in several inactivation-less mutants. In the upcoming period we plan to delineate the mechanism for this transmembrane allosteric control of channel activity. In our first aim, we will systematically identify residues that participate in the mechanism, i.e. residues that “sense” and “couple” both binding phenomena and mediate the allosteric response. We will implement an NMR chemical shift based strategy to identify likely candidates. To confirm the key role of candidate sites, we will manipulate the strength of the coupling through mutation at these sites. The functional hallmark of allostery, modulation of ligands’ affinities through binding of another, distal ligand, will be probed by NMR to quantitatively assess the impact of mutation on coupling. In our second aim, we will determine the structure of the Activated state and contrast key interactions involving the allosteric participants in the Activated state vs the Deactivated (Resting) and Inactivated states, to test hypotheses about the molecular basis for allostery. The Activated state is the only state that transmits ions, and is the key metastable intermediate of allosteric response. The structure of the wild type activated channel in bilayers has been elusive. In contrast to other kinds of studies, our SSNMR studies are done on hydrated, wild type channels in bilayers; the pH and ion concentrations are freely varied. In the previous grant period, we identified conditions for preparing the Activated state. In our third aim we will characterize dynamic exchange processes in the Activated state in order to obtain insights into spontaneous Inactivation. We will use recently developed rotating frame solid state NMR pulse sequences that allow measurement at numerous sites, minimizing unwanted coherent evolution of the spins. We will contrast the conformational dynamics of the Activated state in hydrated bilayers to other states of the system (Deactivated, Inactivated). By comparing the exchange timescales and amplitudes to those expected from MD-based models of activation coupled inactivation we will test a variety of mechanisms for allosteric inactivation of ion channels.

抽象的 钾通道控制人类和病原体的许多信号传导过程。基本上全部 由于跨膜变构，K+通道在打开后会自发失活 控制平均开放时间的过程。失活调节许多重要通道的功能 和药物靶点：例如，神经元利用 K+ 通道失活来调节其放电频率，以及 人类心脏通道的失活对心脏计时有很大影响。我们最近的工作提供了 有证据表明 KcsA 中 C 型失活的分子基础是跨膜变构偶联，其中 细胞内激活门的打开导致细胞外选择性过滤器失去对 K+ 的亲和力。我们 表明这种跨膜变构耦合在双层的野生型通道中很强，而在 几个失活较少的突变体。在接下来的一段时间内，我们计划为此制定机制 通道活性的跨膜变构控制。在我们的第一个目标中，我们将系统地识别残留物 参与该机制，即“感知”和“耦合”结合现象并介导 变构反应。我们将实施基于核磁共振化学位移的策略来识别可能的候选者。到 确认候选位点的关键作用后，我们将通过突变来操纵耦合的强度 这些网站。变构的功能标志，通过与另一个配体的结合来调节配体的亲和力， 远端配体将通过 NMR 进行探测，以定量评估突变对偶联的影响。在我们的第二个 目标，我们将确定激活状态的结构并对比涉及变构的关键相互作用 处于激活状态与停用（休息）和失活状态的参与者，以测试有关的假设 变构的分子基础。激活状态是唯一传输离子的状态，是关键 变构反应的亚稳态中间体。双层中野生型激活通道的结构 一直难以捉摸。与其他类型的研究相比，我们的 SSNMR 研究是在水合野生型上进行的 双层通道； pH 值和离子浓度可自由变化。在上一个资助期内，我们确定了 准备激活状态的条件。在我们的第三个目标中，我们将描述动态交换过程的特征 处于激活状态，以便深入了解自发失活。我们将使用最近开发的 旋转框架固态核磁共振脉冲序列，允许在多个地点进行测量，最大限度地减少 不必要的自旋相干演化。我们将对比激活状态的构象动力学 水合双层到系统的其他状态（失活、失活）。通过比较兑换 时间尺度和振幅与基于 MD 的激活耦合失活模型的预期一致，我们将 测试离子通道变构失活的多种机制。