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：
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
项目类别：
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

项目摘要

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.

抽象的钾通道控制着人类和病原体的众多信号传导过程。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，以及我们最近的研究表明，人类心脏通道的失活对心脏计时有很大影响。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。表明这种跨膜变构耦合在双层的野生型通道中很强，而在在接下来的一段时间内，我们计划描述一些无失活突变体的机制。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基参与该机制，即“感知”和“耦合”结合现象并介导我们将实施基于 NMR 化学位移的策略来识别可能的候选者。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位点的功能标志，通过结合另一个配体的亲和力来调节，在我们的第二个研究中，将通过 NMR 探测远端配体，以定量评估突变对偶联的影响。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）和停用状态的参与者，以测试有关的假设变构的分子基础激活态是传输离子的唯一状态，也是关键。双层中野生型激活通道的结构具有变构反应的亚稳态中间体。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。双层中的通道；pH 值和离子浓度可以自由变化。在我们的第三个目标中，我们将描述动态交换过程的特征。为了深入了解自发失活，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，最大限度地减少我们将对比激活态的构象动力学。通过比较交换，水合双层与系统的其他状态（失活、失活）。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。