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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：
10021668
负责人：
ANN E MCDERMOTT
金额：
$ 35.98万
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 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10021668
关键词：
Affect Affinity Amino Acids Arrhythmia Behavior Binding Cardiac Cell Signaling Process Chemicals Communicable Diseases Coupled Coupling Data Disease Distal Drug Targeting Event Evolution Exhibits Freezing Frequencies Grant Health Heart Human Hydration status Investigation Ion Channel Ions Laboratories Length Life Ligand Binding Ligands Long QT Syndrome Malignant Neoplasms Measurement Mediating Medical Membrane Membrane Potentials Methods Modeling Molecular Molecular Conformation Mutagenesis Mutation Nature Nervous System Physiology Nervous system structure Neurons Participant Pharmaceutical Preparations Physiologic pulse Physiological Play Potassium Channel Process Property Protein Region Regulation Regulatory Element Rest Role Signal Transduction Site Structure Study models Syndrome System Testing Thermodynamics Time Work base design experience experimental study extracellular human pathogen insight mutant nanosecond potassium ion receptor response sensor solid state nuclear magnetic resonance tool

项目摘要

Abstract Potassium channels control numerous signaling processes for humans and pathogens. Essentially all characterized K+ channels inactivate spontaneously after opening, due to a transmembrane allosteric process that acts to control mean open time. Inactivation modulates function for many important channels and drug targets: for example, neurons use K+ channel inactivation to modulate their firing frequency, and inactivation in the channels of the human heart has strong effects on heart timing. Our recent work provided evidence that the molecular basis of C type inactivation in KcsA is transmembrane allosteric coupling, where opening of the intracellular activation gate causes the extracellular selectivity filter to lose its affinity for K+. We showed that this transmembrane allosteric coupling is strong in the wild type channel in bilayers and absent in several inactivation-less mutants. In the upcoming period we plan to delineate the mechanism for this transmembrane allosteric control of channel activity. In our first aim, we will systematically identify residues that participate in the mechanism, i.e. residues that “sense” and “couple” both binding phenomena and mediate the allosteric response. We will implement an NMR chemical shift based strategy to identify likely candidates. To confirm the key role of candidate sites, we will manipulate the strength of the coupling through mutation at these sites. The functional hallmark of allostery, modulation of ligands’ affinities through binding of another, distal ligand, will be probed by NMR to quantitatively assess the impact of mutation on coupling. In our second aim, we will determine the structure of the Activated state and contrast key interactions involving the allosteric participants in the Activated state vs the Deactivated (Resting) and Inactivated states, to test hypotheses about the molecular basis for allostery. The Activated state is the only state that transmits ions, and is the key metastable intermediate of allosteric response. The structure of the wild type activated channel in bilayers has been elusive. In contrast to other kinds of studies, our SSNMR studies are done on hydrated, wild type channels in bilayers; the pH and ion concentrations are freely varied. In the previous grant period, we identified conditions for preparing the Activated state. In our third aim we will characterize dynamic exchange processes in the Activated state in order to obtain insights into spontaneous Inactivation. We will use recently developed rotating frame solid state NMR pulse sequences that allow measurement at numerous sites, minimizing unwanted coherent evolution of the spins. We will contrast the conformational dynamics of the Activated state in hydrated bilayers to other states of the system (Deactivated, Inactivated). By comparing the exchange timescales and amplitudes to those expected from MD-based models of activation coupled inactivation we will test a variety of mechanisms for allosteric inactivation of ion channels.

摘要