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

基本信息

批准号：
10659941
负责人：
ANN E MCDERMOTT
金额：
$ 47.18万
依托单位：
COLUMBIA UNIV NEW YORK MORNINGSIDE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2027-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10659941
关键词：
Acceleration Affinity Amino Acid Motifs Antibiotics Architecture Arrhythmia Bacteria Binding Binding Sites Biophysics Cardiac Chemicals Collection Complex Coupling Data Data Analyses Disease Drug Targeting Electrophysiology (science)Event Exhibits Frequencies Functional disorder Funding Grant Health Heart Homologous Gene Human Hydration status Ion Channel Ions Kinetics Laboratories Lead Length Life Ligand Binding Ligands Location Long QT Syndrome Measurement Measures Medical Membrane Membrane Potentials Methods Modeling Molecular Molecular Conformation Molecular Probes Mutation NMR Spectroscopy Nervous System Neurons Pathway interactions Pharmaceutical Preparations Physiologic pulse Potassium Channel Process Property Protein Region Protons Regulation Regulatory Element Relaxation Reporting Research Role Sequence Analysis Signal Transduction Source Structure Testing Thermodynamics Time base carbonyl group experimental study extracellular insight interest methicillin resistant Staphylococcus aureus millisecond molecular dynamics mutant pathogen pathogenic bacteria proteoliposomes response sensor small molecule solid state nuclear magnetic resonance voltage gated channel

项目摘要

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.

钾通道基本上控制着人类和病原体的膜电位和信号传导过程。由于跨膜变构过程，K+通道在打开后失活，这似乎是激活速度缓慢，并且涉及孔域中选择性过滤器附近的残基（c 型失活）。激活状态是唯一的导电物质，并且是亚稳态的，失活控制意味着开放时间和从而调节功能对于许多重要的通道和药物靶标，例如神经元使用 K+ 通道。调节其发射频率的失活动力学，以及人类心脏通道中的失活动力学我们的研究提供了 K+ 通道 KcsA 的分子基础证据。 c型失活是激活门（H+与细胞内pH值结合）之间的跨膜变构耦合在最近的努力中，我们已经描述了失活门（K+从细胞外选择性过滤器释放）。通过识别发挥重要作用的残基来跨膜变构控制通道活性的机制在使用核磁共振化学位移和突变的变构反应中，我们表明激活和开放直接导致。对于野生型，选择性过滤器中 K+ 损失，但对于失活减少的突变体则不然，因为热力学耦合我们的研究使用固态 NMR 测量全长野生型。水合蛋白脂质体中的通道并提供原子进入结构以及动力学和因此，上一时期的研究为变构偶联的假设提供了支持。激活和失活之间的关系是失活和通道计时的基础，这些研究也为这一点提供了支持。 “热点”的具体身份（目标 1）在接下来的一段时间内，最终的测试将基于突变体。加速失活，从某种意义上说，这些突变体增强了“计时”功能，而不是废除了它。这些失活更快的突变体也引起人们的兴趣，因为它们与真核通道有相似之处或相似之处（目标 2）我们计划探索通道激活开放状态的构象动力学。最近开发了核磁共振方法，以识别自发转化为早期特定失活中间体。上一时期开发的方法使我们能够对关键羰基和芳香基团的动力学进行研究。将利用固态核磁共振方法灵敏度方面的最新突破来进行控制测试动力学和功能之间已建立的关系，例如通过分子动力学模拟。（目标3）最后，在Ktr这个已被确定为许多病原菌药物靶点的相关通道中，我们计划我们还将阐明配体通道相互作用，确定有前途的先导化合物的结合位置。表征失活机制并确定变构、失活和功能的后果更清楚地了解这些跨膜示例中的热力学和动力学。变构可能会澄清广泛的生物物理原理，以及对小分子的具体见解疾病相关通道功能的调节剂。