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.

钾通道控制人类和病原体的膜电位和信号传导过程。由于跨膜变构过程而打开后的K+通道灭活后灭活的表征激活的结果缓慢，并涉及孔域中选择性过滤器附近的残基。活化状态是唯一的导电物种，并且是可稳定的，灭活控制是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。灭活动力学调节其发射频率和人类心脏通道中的灭活动力学对我们的研究有很大的影响。 C型灭活是激活门之间的跨膜变构耦合（H+与细胞内pH结合传感器）和灭活门（从细胞外选择过滤器释放K+）。通过识别起重要作用的居民，对通道活性的跨膜变构控制机制在使用NMR化学移位和突变的变构反应中。野生型的选择性滤波器中的K+损失，但没有用于灭活的突变体，热力学耦合在开放和K+亲和力之间，我们的研究使用固态NMR测量水合蛋白质脂质体中的通道，并提供对结构的原子访问以及动力学和动力学因此，配体结合的热力学。激活和灭活是失活的基础和渠道时机。 “热点”的特定身份。从某种意义上说，在灭活中加速了，从而增加了时机“功能而不是废除它。这些更快的速度也具有与真核通道相似的相似之处快速灭活（AIM 2）我们计划最近开发了NMR方法，以识别自发转化向早期失活的中间。在上一个时期开发的方法使我们能够对关键羰基和芳香族组的动态进行研究。将利用固态NMR方法的敏感性的最新突破，以便可以执行控件测试动力学与功能之间的假设关系，例如通过分子动力学模拟。（AIM 3）最后，在KTR中，与许多致病性Bactoria的药物目标相关的渠道，我们计划澄清配体通道相互作用确定有希望的铅化合物的结合位置表征失活机制，并确定分配，灭活和功能的肯定配体结合了对这些跨膜的热力学和动力学的更清晰的理解变构可能澄清阐明广泛的生物物理原理，以及对小分子的特异性见解与疾病相关的通道功能的调节剂。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Investigation of slow molecular dynamics using R-CODEX.

DOI：
10.1016/j.jmr.2012.05.019
发表时间：
2012-09
期刊：
JOURNAL OF MAGNETIC RESONANCE
影响因子：
2.2
作者：
Li, Wenbo;McDermott, Ann
通讯作者：
McDermott, Ann

Contribution of protein conformational heterogeneity to NMR lineshapes at cryogenic temperatures.

低温下蛋白质构象异质性对 NMR 线形的贡献。

DOI：
10.1073/pnas.2301053120
发表时间：
2024
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Yi,Xu;Fritzsching,KeithJ;Rogawski,Rivkah;Xu,Yunyao;McDermott,AnnE
通讯作者：
McDermott,AnnE

Protein linewidth and solvent dynamics in frozen solution NMR.