Structural and Functional Studies of Potassium Channels by Solid State NMR

描述（由申请人提供）：所有研究的K+通道在打开后自发失活，包括模型通道和决定人类心脏时间的hERG通道。失活通过确定平均打开时间和通道重新打开前的延迟来控制通道信号，但其分子基础仍然存在争议，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：从选择性过滤器释放K+离子是失活的必要步骤吗？此外，由于跨膜变构耦合，它被假设自发发生：细胞内H+触发的内部跨膜螺旋TM2的变化产生导电激活状态，但这会产生冲突，破坏细胞外K+负载选择性过滤器的稳定，并导致其缓慢衰减到K+耗尽状态。质子激活通道KcsA提供了一个独特的机会来详细了解这种失活过程。与x射线衍射或溶液核磁共振研究相反，拟议的固态核磁共振研究将在水合膜双层中的全长KcsA上进行，使用野生型或突变体和不同的缓冲条件；因此，在电生理学中识别的功能物种可以方便地制备核磁共振。失活的关键特征（突变依赖性、动力学和[K+]依赖性）证实了低pH核磁共振检测到的物种是失活状态。失活突变体的改变将用于进一步测试K+释放是否对失活至关重要。有几种未观察到失活，pH值3-5的优势种为活化态。对其他失活发生定量，优势种在pH 3-5是失活状态。比较各种突变体，失活（通过电生理学）和K+消耗（通过核磁共振）之间的相关性将为我们的假设提供一个明确的测试。野生型和E71A的初步研究为这一假设提供了强有力的支持。静息、活化和失活的电生理相互转化速率将与核磁共振的K+释放速率进行比较。我们最近的4D核磁共振数据允许全光谱分配，并首次表明变构耦合在两个方向上起作用：不仅pH传感器的质子化导致高环境[K+]下的K+离子释放，而且K+离子在低环境[K+]下的提取导致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。核磁共振滴定将允许定量描述变构偶联，澄清双分子层和关键氨基酸的作用，使用最近描述的偶联受损突变体，其中去除选择性过滤器和TM2铰链之间的大块侧链。迄今为止，失活状态的高分辨率结构一直难以捉摸。高质量的失活状态核磁共振光谱为完整双层结构的确定提供了极好的机会，如本文所提出的。

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.