Structural and Functional Studies of Potassium Channels by Solid State NMR

钾通道控制着人类和病原体的膜电位和信号过程。基本上都是 由于跨膜变构过程，特征的K+通道开放后失活 缓慢的活化结果，涉及孔域中选择性过滤器附近的残留物(C型失活)。自.以来 激活状态是唯一的导电物种，并且是亚稳态，失活控制意味着打开时间和 从而调节功能 许多重要渠道和毒品目标。例如，神经元使用K+通道 调节其放电频率的失活动力学，以及人体心脏通道中的失活动力学 对心脏计时有很强的影响。我们的研究为K+通道KCsA的分子基础提供了证据 C型失活是指激活门之间的跨膜变构偶联(H+结合到细胞内的pH 传感器)和失活门(从细胞外选择性过滤器释放K+)。在最近的努力中，我们描绘了 跨膜变构通过识别起重要作用的残基来控制通道活性的机制 在变构反应中使用核磁共振化学位移和突变。我们证明了激活和开放直接导致 K+在野生型选择性过滤器中的损失，但对于失活减少突变体，热力学耦合不是这样 开放与K+之间的亲和力降低。我们的研究使用的是全长野生型的固态核磁共振测量 水合蛋白脂质体中的通道，并提供原子化的结构途径，以及动力学和 配基结合的热力学。因此，上一阶段的研究支持了变构偶联的假设 激活和失活之间的关系是失活和通道定时的基础。这些研究也支持了 “热点”的具体身份。(目标1)在即将到来的时期，最终的测试将基于符合以下条件的突变 加速失活，在某种意义上说，这些突变体增加了“计时”功能，而不是取消了它。许多. 这些失活速度更快的突变体也令人感兴趣，因为它们与真核细胞通道有相似之处或相似之处 快速失活。(目标2)我们计划探索激活的通道开放状态的构象动力学 最近开发的核磁共振方法，以确定自发转化为早期失活的中间体。特定的 上一阶段开发的方法使我们能够对关键的羰基和芳香基的动力学进行研究。 将利用固体核磁共振方法在灵敏度方面的最新突破，以便进行控制 以测试动力学和功能之间的假设关系，例如从分子动力学模拟。 (目标3)最后，在Ktr，一个已被确定为许多致病细菌的药物靶标的相关渠道，我们计划 阐明配体-通道相互作用决定了有希望的先导化合物的结合位置。我们还将 表征失活机制，并确定以下情况对变构、失活和功能的影响 配体结合在一起。更清楚地了解这些跨膜样品中的热力学和动力学 变构很可能导致对广泛的生物物理原理的澄清，以及对小分子的具体见解。 疾病相关通道功能的调节器。

Potassium channels control membrane potential and signaling processes for humans and pathogens. Essentially all characterized K+ channels inactivate after opening due to a transmembrane allosteric process that appears to be a slow result of activation and involves residues near the selectivity filter in the pore domain (c-type inactivation). Since the activated state is the only conductive species, and is metastable, inactivation controls mean open time and thereby modulates function for many important channels and drug targets. For example, neurons use K+ channel inactivation kinetics to modulate their firing frequency, and inactivation kinetics in the channels of the human heart have strong effects on heart timing. Our research provided evidence in the K+ channel KcsA that the molecular basis of c-type inactivation is transmembrane allosteric coupling between the activation gate (H+ binding to the intracellular pH sensor) and the inactivation gate (K+ release from the extracellular selectivity filter). In recent efforts, we have delineated the mechanism for transmembrane allosteric control of channel activity by identifying residues that serve important roles in the allosteric response using NMR chemical shifts and mutation. We showed that activation and opening directly lead to K+ loss in the selectivity filter for wild type, but not for inactivation-reduced mutants, for which thermodynamic coupling between opening and K+ affinity is reduced. Our studies use solid state NMR measurements on full-length wild-type channels in hydrated proteoliposomes and offer atomistic access to structure, as well as the dynamics and thermodynamics of ligand binding. Thus, studies in the last period offer support for the hypothesis that allosteric coupling between activation and inactivation is the basis for inactivation and channel timing. The studies also give support for the specific identities of the “hotspots”. (Aim 1) In the upcoming period, a definitive test will be based on mutants that are accelerated in inactivation, in the sense that these mutants increase the “timing” function rather than abolish it. Many of these faster-inactivating mutants are also of interest because of similarities or analogies to eukaryotic channels that are fast-inactivating. (Aim 2) We plan to probe the conformational dynamics of the activated open state of the channel with recently developed NMR methods, to identify spontaneous conversion to early intermediates of inactivation. Specific methods developed in the last period allow us to carry out studies of the dynamics of key carbonyl and aromatic groups. Recent breakthroughs in the sensitivity of solid state NMR methods will be harnessed so that controls can be performed to test hypothesized relationships between dynamics and function, for example from molecular dynamics simulations. (Aim 3) Finally, in Ktr, a related channel that has been identified as a drug target for many pathogenic bacteria, we plan to clarify ligand-channel interactions determining the binding location of promising lead compounds. We will also characterize the inactivation mechanism and determine the consequence to allostery, inactivation and function when ligands bind. A clearer understanding of the thermodynamics and dynamics in these exemplars of transmembrane allostery is likely to lead to clarification of broad biophysical principles, as well as specific insights into small molecule modulators of disease-related channel function.