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
• 负责人: ANN E MCDERMOTT
• 金额: $ 36.05万
• 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/10224775
• 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众多信号传导过程。 开放后自发性地表征了K+通道，应有的跨膜变构 用于控制未开放时间的过程。 和药物靶标：例如，神经元使用K+通道灭活来调节其发射频率 人心渠道中的失活对我们最近的工作提供了强烈的影响。 CSA中C类型灭活的分子基础的证据是跨膜变构耦合，是 细胞内激活门的打开会导致细胞外选择性滤波器失去K+ 显示了双层中野生型通道中的跨膜变构耦合，而在 几个无活化的突变体。 跨膜变构对信道活动的控制。 参与机制 变构响应 确认候选人的关键作用，我们将操纵耦合突变的强度 这些位点。 NMR将探测远端配体，以评估突变对耦合的影响。 目的，我们将确定激活状态的结构和对比键相互作用的变形 激活状态的参与者与停用（静止）和灭活状态的参与者，以检验有关的假设 活化状态的分子基础是传输离子的状态，是钥匙 异构响应的亚稳态。 与其他研究相比，我们的SSNMR研究是难以捉摸的。 双层中的渠道在上一批赠款期间自由变化。 在我们的第三个目标中，我们将表征动态交换过程 在激活状态下，我们将使用最近开发的灭活状态。 旋转框架固态NMR脉冲序列，可以在许多坐着时进行测量，从而最大程度地减少 旋转的不良相干演变。 在系统的其他状态下，将双层造成的水合（通过灭活，灭活）。 从基于MD的激活耦合模型中预期的时间表和幅度。 测试各种机制，以使离子通道的变构失活。