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）：所有研究的 K+ 通道（包括模型通道和决定人类心脏计时的 hERG 通道）打开后会自发发生失活。失活通过确定平均开放时间和重新开放之前的延迟来控制通道信号传导，但其分子基础仍然存在争议，并提出了几种模型。我们建议澄清区分这些模型的一个关键方面：从选择性过滤器中释放 K+ 离子是失活的重要步骤吗？此外，假设由于跨膜变构耦合而自发发生：细胞内 H+ 触发内跨膜螺旋 TM2 的变化，产生导电激活状态，但这会产生冲突，使细胞外 K+ 负载的选择性过滤器不稳定，并导致其缓慢流动。衰变至 K+ 耗尽状态。 KcsA 是一种质子激活通道，为详细了解这种失活过程提供了独特的机会。与 X 射线衍射或溶液 NMR 研究相反，拟议的固态 NMR 研究将使用野生型或突变体以及不同的缓冲条件对水合膜双层中的全长 KcsA 进行；因此，可以方便地制备电生理学中鉴定的功能物质用于核磁共振。失活的关键特征（突变依赖性、动力学和 [K+] 依赖性）证实低 pH NMR 检测到的物种处于失活状态。失活发生改变的突变体将用于进一步测试 K+ 释放是否对失活至关重要。对于几种没有观察到失活，并且 pH 3-5 时的优势物种是激活状态。对于其他物质，失活是定量发生的，pH 3-5 时的优势物种是失活状态。比较各种突变体，失活（通过电生理学）和 K+ 消耗（通过 NMR）之间的相关性将为我们的假设提供清晰的检验。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。电生理学中的静息、激活和失活的相互转化率将与 NMR 中的 K+ 释放率进行比较。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两个方向上起作用：pH 传感器的质子化不仅会在高环境 [K+] 下导致 K+ 离子释放，而且还会在高环境 [K+] 下导致 K+ 离子提取。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+ 通道的新机制。 NMR 滴定将允许使用最近描述的耦合受损突变体（其中选择性过滤器和 TM2 铰链之间的庞大侧链被去除）对变构耦合进行定量描述，澄清双层和关键氨基酸的作用。迄今为止，失活状态的高分辨率结构一直难以捉摸。如本文所提出的，失活状态的高质量核磁共振谱为完整双层的结构测定提供了极好的机会。