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:
- 金额:$ 35.98万
- 依托单位:
- 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:
- 关键词:AffectAffinityAmino AcidsArrhythmiaBehaviorBindingCardiacCell Signaling ProcessChemicalsCommunicable DiseasesCoupledCouplingDataDiseaseDistalDrug TargetingEventEvolutionExhibitsFreezingFrequenciesGrantHealthHeartHumanHydration statusInvestigationIon ChannelIonsLaboratoriesLengthLifeLigand BindingLigandsLong QT SyndromeMalignant NeoplasmsMeasurementMediatingMedicalMembraneMembrane PotentialsMethodsModelingMolecularMolecular ConformationMutagenesisMutationNatureNervous System PhysiologyNervous system structureNeuronsParticipantPharmaceutical PreparationsPhysiologic pulsePhysiologicalPlayPotassium ChannelProcessPropertyProtein RegionRegulationRegulatory ElementRestRoleSignal TransductionSiteStructureStudy modelsSyndromeSystemTestingThermodynamicsTimeWorkbasedesignexperienceexperimental studyextracellularhuman pathogeninsightmutantnanosecondpotassium ionreceptorresponsesensorsolid state nuclear magnetic resonancetool
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
钾通道控制着人类和病原体的许多信号传导过程。基本上所有
特征性K+通道在开放后自发地,由于跨膜变构,
用于控制平均开放时间的过程。失活调节许多重要通道的功能
和药物靶点:例如,神经元使用K+通道失活来调节其放电频率,
人心脏通道中的失活对心脏计时具有强烈的影响。我们最近的工作提供了
证明KcsA中C型失活的分子基础是跨膜变构偶联,其中
细胞内激活门的打开导致细胞外选择性过滤器失去其对K+的亲和力。我们
表明这种跨膜变构偶联在双层的野生型通道中很强,而在
几种无失活突变体在今后一段时期,我们计划为此制定机制,
通道活性的跨膜变构控制。在我们的第一个目标中,我们将系统地鉴定
参与机制,即“感测”和“偶联”结合现象并介导结合的残基。
变构反应我们将实施基于NMR化学位移的策略来识别可能的候选者。到
为了确认候选位点的关键作用,我们将通过突变来操纵偶联的强度,
这些网站。变构的功能标志是通过结合另一种配体来调节配体的亲和力,
通过NMR探测远端配体,以定量评估突变对偶联的影响。在我们的第二
目的,我们将确定激活状态的结构,并对比涉及变构的关键相互作用。
参与者在激活状态与停用(休息)和停用状态,以测试假设
变构的分子基础激活状态是唯一能传输离子的状态,也是关键。
变构反应的亚稳态中间体。双层中野生型激活通道的结构具有
难以捉摸与其他类型的研究相比,我们的SSNMR研究是在水合的野生型
通道的双层; pH值和离子浓度自由变化。在上一个补助期,我们确定了
准备激活状态的条件。在我们的第三个目标,我们将描述动态交换过程
在激活状态,以获得对自发失活的见解。我们将使用最近开发的
旋转帧固态NMR脉冲序列,允许在多个位置进行测量,
自旋的不必要的相干演化。我们将对比活化态的构象动力学
在水合的双层中,系统的其他状态(失活、失活)。通过比较交易所
时间尺度和幅度的预期从MD的激活耦合失活模型,我们将
测试离子通道变构失活的多种机制。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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ANN E MCDERMOTT其他文献
ANN E MCDERMOTT的其他文献
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{{ truncateString('ANN E MCDERMOTT', 18)}}的其他基金
HIGH FIELD/HIGH FREQUENCY ESR FOR STUDYING DNP IN BIOMEMBRANES
用于研究生物膜中 DNP 的高场/高频 ESR
- 批准号:
8364114 - 财政年份:2011
- 资助金额:
$ 35.98万 - 项目类别:
DYNAMIC NUCLEAR POLARIZATION SOLID STATE NMR SPECTROMETER FOR BIOMOLECULAR STUDIE
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- 批准号:
7839443 - 财政年份:2010
- 资助金额:
$ 35.98万 - 项目类别:
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
- 批准号:
10460945 - 财政年份:2009
- 资助金额:
$ 35.98万 - 项目类别:
Structural and Functional Studies of Channels and Pumps by Solid State NMR
通过固态核磁共振研究通道和泵的结构和功能
- 批准号:
8325732 - 财政年份:2009
- 资助金额:
$ 35.98万 - 项目类别:
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
- 批准号:
8760232 - 财政年份:2009
- 资助金额:
$ 35.98万 - 项目类别:
Structural and Functional Studies of Channels and Pumps by Solid State NMR
通过固态核磁共振研究通道和泵的结构和功能
- 批准号:
7941916 - 财政年份:2009
- 资助金额:
$ 35.98万 - 项目类别:
Structural and Functional Studies of Channels and Pumps by Solid State NMR
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- 批准号:
8142738 - 财政年份:2009
- 资助金额:
$ 35.98万 - 项目类别:
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
- 批准号:
9117619 - 财政年份:2009
- 资助金额:
$ 35.98万 - 项目类别:
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
- 批准号:
10224775 - 财政年份:2009
- 资助金额:
$ 35.98万 - 项目类别:
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
- 批准号:
10659941 - 财政年份:2009
- 资助金额:
$ 35.98万 - 项目类别:
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