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
• 负责人: ANN E MCDERMOTT
• 金额: $ 27.67万
• 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 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8760232
• 关键词: Affinity; Amino Acids; Arrhythmia; Behavior; Binding; Binding Sites; Buffers; Cardiac; Characteristics; Coupled; Coupling; Crystallography; Data; Dependence; Electrophysiology (science); Environment; Event; Fingerprint; Goals; Grant; Heart; Human; Ion Channel; Ions; Kinetics; Length; Life; Long QT Syndrome; Measurement; Measures; Medical; Membrane; Methods; Modeling; Molecular; Molecular Conformation; Monitor; Motion; Mutation; Nervous system structure; Organism; Pharmaceutical Preparations; Physiological; Physiology; Population; Potassium Channel; Process; Property; Proteins; Protons; Resolution; Rest; Roentgen Rays; Role; Signal Transduction; Site; Solutions; Structural Models; Structure; Testing; Time; Titrations; base; enthalpy; extracellular; mutant; novel; protonation; public health relevance; research study; sensor; solid state nuclear magnetic resonance; stoichiometry; three dimensional structure

DESCRIPTION (provided by applicant): Inactivation occurs spontaneously after opening in all studied K+ channels, including model channels and the hERG channel that determines timing of the human heart. Inactivation controls channel signaling by determining mean open times and the delay before they can be re-opened, yet its molecular basis remains controversial, with several models proposed. We propose to clarify a key aspect distinguishing these models: is K+ ion release from the selectivity filter an essential step in inactivation? Also, it is hypothesizedto occur spontaneously because of transmembrane allosteric coupling: intracellular H+-triggered changes in the inner transmembrane helix, TM2, produce the conductive Activated state, but this creates clashes that destabilize the extracellular K+ loaded selectivity filter, and cause it o slowly decay to the K+ depleted state. KcsA, a proton-activated channel, provides a unique opportunity to understand this inactivation process in detail. In contrast to the X-ray diffractionor solution NMR studies, the proposed Solid State NMR studies will be performed on full- length KcsA in hydrated membrane bilayers, using wild type or mutants and varied buffer conditions; hence functional species identified in electrophysiology can be conveniently prepared for NMR. Key signatures for inactivation (mutation dependences, kinetics, and [K+] dependence) confirm that the low pH NMR-detected species is the Inactivated state. Mutants altered in inactivation will be used to further test whether K+ release is essential to inactivation. For several no inactivation is observed, and the dominant species at pH 3-5 is the Activated state. For others inactivation occurs quantitatively, and the dominant species at pH 3-5 is the Inactivated state. Comparing various mutants, correlation between inactivation (by electrophysiology) and K+ depletion (by NMR) will provide a clear test of our hypothesis. An initial study of wild type and E71A provides strong support for this hypothesis. Interconversion rates of the Resting, Activated and Inactivated from electrophysiology will be compared with the K+ release rates from NMR. Our recent 4D NMR data allow full spectral assignments, and show for the first time that the allosteric coupling operates in both directions: not only does protonation of the pH sensor cause K+ ion release at high ambient [K+], but also K+ ion extraction at low [K+] causes pH sensor protonation and opening of TM2 at neutral pH, which represents a novel mechanism for opening a K+ channel. NMR titrations will allow quantitative description of the allosteric coupling, clarifying of the role of the bilayer, and of key amino acids, using recently described coupling-impaired mutants, where bulky sidechains between the selectivity filter and the hinge of TM2 are removed. The high-resolution structure for the inactivated state has been elusive to date. High-quality NMR spectra of the inactivated state provide an excellent opportunity for structure determination in intact bilayers, as proposed herein.

描述（由申请人提供）：在所有研究的K+通道中打开后，灭活是自发发生的，包括模型通道和决定人心脏时间的HERG通道。灭活方法通过确定平均开放时间和可以重新开放之前的延迟来控制信号传导，但提出了几种模型，但其分子基础仍然存在争议。我们建议阐明区分这些模型的关键方面：K+离子与选择性过滤的释放是否是失活的重要步骤？而且，假设是由于跨膜变构耦合而自发发生的：细胞内H+触发的内膜内膜螺旋中TM2的变化会产生导电性活化状态，但这会产生冲突，使其破坏了细胞外K+负载的选择性滤镜，并缓慢地导致IT o，并缓慢地导致其稳定。衰减到K+耗尽状态。 KCSA是一种质子激活的渠道，提供了一个独特的机会，可以详细了解这种失活过程。与X射线衍射剂溶液NMR研究相反，提出的固态NMR研究将使用野生型或突变体以及各种缓冲液条件在水合膜双层中的全长KCSA上进行。因此，可以方便地为NMR制备在电生理学中鉴定出的功能性物种。失活的关键签名（突变依赖，动力学和[K+]依赖性）确认低pH NMR检测的物种是灭活状态。灭活中改变的突变体将用于进一步测试K+释放是否对于灭活至关重要。对于几个没有灭活的灭活，pH 3-5处的主要物种是活化状态。对于其他人来说，灭活是定量发生的，pH 3-5处的主要物种是灭活状态。比较各种突变体，失活（通过电生理学）和K+耗竭（通过NMR）之间的相关性将对我们的假设进行清晰的检验。对野生型和E71A的初步研究为这一假设提供了强有力的支持。将静止，激活和从电生理灭活的静止转换速率与NMR的K+释放速率进行比较。我们最近的4D NMR数据允许完整的光谱分配，并首次表明变构耦合在这两个方向上运行：pH传感器的质子化不仅会在高环境[K+]处引起K+离子释放，还会在K+离子提取处释放K+离子。低[K+]会导致pH传感器质子化并在中性pH下打开TM2，这代表了打开K+通道的一种新型机制。 NMR滴定将允许使用最近描述的耦合损伤突变体的变构耦合，澄清双层的作用和关键氨基酸的作用，其中选择性滤波器和TM2的铰链之间的笨重的侧侧被移除。灭活状态的高分辨率结构迄今难以捉摸。如本文所建议的那样，灭活状态的高质量NMR光谱为完整双层的结构确定提供了绝佳的机会。