Structural and Functional Studies of Potassium Channels by Solid State NMR

描述(由申请人提供)：在所有研究的K+通道，包括模型通道和决定人体心脏计时的HERG通道开放后，失活会自发发生。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，但其分子基础仍然存在争议，提出了几个模型。我们建议澄清区分这些模型的一个关键方面：K+离子从选择性过滤器中释放是失活的必要步骤吗？此外，它被认为是由于跨膜变构偶联而自发发生的：细胞内H+引发的内跨膜螺旋TM2的变化，产生传导激活状态，但这会产生冲突，破坏细胞外K+负载的选择性过滤器的稳定性，并导致它缓慢衰退到K+耗尽状态。KCSA，一个质子激活的通道，为详细了解这种失活过程提供了一个独特的机会。与X射线衍射法或溶液核磁共振研究不同的是，所提出的固体核磁共振研究将使用野生型或突变体和不同的缓冲条件，对水合膜双层中的全长KCSA进行研究；因此，可以方便地制备电生理学中鉴定的功能物种用于核磁共振。失活的关键信号(突变依赖、动力学和[K+]依赖)证实低pH核磁共振检测到的物种是失活状态。在失活中改变的突变体将被用来进一步测试K+释放是否是失活所必需的。在pH 3-5范围内，NO主要以活化状态存在。另一些则是定量失活，在pH值为3-5时，优势物种为失活状态。比较各种突变体，失活(通过电生理学)和K+耗尽(通过核磁共振)之间的相关性将提供一个明确的测试我们的假设。对野生型和E71a的初步研究为这一假说提供了强有力的支持。电生理的静息、激活和失活的相互转化率将与核磁共振的K+释放率进行比较。我们最近的4D核磁共振数据允许全光谱指认，并首次表明变构耦合是双向的：不仅pH传感器的质子化导致高环境[K+]下K+离子的释放，而且低[K+]下的K+离子提取导致pH传感器质子化和中性pH下TM2的打开，这代表了一种打开K+通道的新机制。核磁共振滴定将允许定量描述变构偶联，澄清双层的作用，以及关键氨基酸的作用，使用最近描述的偶联受损突变体，其中选择性过滤器和TM2铰链之间的笨重侧链被移除。到目前为止，失活状态的高分辨率结构一直难以捉摸。如本文所建议的，失活状态的高质量的核磁共振谱为确定完整双层的结构提供了极好的机会。

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.