Structural and Functional Studies of Potassium Channels by Solid State NMR

描述（由申请方提供）：在所有研究的K+通道（包括模型通道和决定人类心脏时序的hERG通道）中，在开放后自发发生失活。失活通过确定平均开放时间和通道重新开放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，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：K+离子从选择性过滤器释放失活的一个重要步骤？此外，它被假设为自发发生，因为跨膜变构偶联：细胞内H+触发的内跨膜螺旋TM 2的变化，产生导电的活化状态，但这会产生冲突，使细胞外K+负载的选择性过滤器不稳定，并导致其缓慢衰减到K+耗尽状态。KcsA是一种质子激活的通道，它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NMR研究相反，将使用野生型或突变体和不同的缓冲液条件对水合膜双层中的全长KcsA进行拟定的固态NMR研究;因此，可以方便地制备电生理学中鉴定的功能物质用于NMR。灭活的关键特征（突变依赖性、动力学和[K +]依赖性）证实低pH NMR检测到的物质为灭活状态。失活改变的突变体将用于进一步测试K+释放是否是失活所必需的。对于几种，未观察到失活，并且在pH 3 - 5下的主要物质为活化状态。对于其他人的失活发生定量，在pH 3 - 5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+耗竭（通过NMR）之间的相关性将为我们的假设提供一个明确的测试。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。将来自电生理学的静息、活化和失活的相互转化率与来自NMR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，并首次显示，变构耦合在两个方向上操作：不仅质子化的pH传感器导致K+离子释放在高环境[K +]，但也K+离子提取在低[K +]导致pH传感器质子化和开放的TM 2在中性pH值，这代表了一种新的机制，打开一个K+通道。NMR滴定将允许定量描述的变构耦合，澄清的作用的双层，和关键氨基酸，使用最近描述的耦合受损的突变体，其中庞大的侧链之间的选择性过滤器和铰链的TM2被删除。迄今为止，失活状态的高分辨率结构一直难以捉摸。高品质的NMR光谱的失活状态提供了一个很好的机会，在完整的双层结构测定，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.