Structural determinants of lipid modulation of ligand-gated ion channels
配体门控离子通道脂质调节的结构决定因素
基本信息
- 批准号:10285984
- 负责人:
- 金额:$ 6.64万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2020
- 资助国家:美国
- 起止时间:2020-07-01 至 2022-06-30
- 项目状态:已结题
- 来源:
- 关键词:AddressAffectAffinityAnestheticsAntiepileptic AgentsBindingBinding SitesBiochemicalBiological ModelsChargeChemicalsChemistryDataDependenceDiseaseEnvironmentEpilepsyErwiniaFatty AcidsFoundationsFunctional disorderGasesGoalsHeadHomologous GeneHydrophobicityIon Channel GatingLabelLearningLengthLigandsLightLipid BindingLipidsLiposomesMass Spectrum AnalysisMeasuresMediatingMembraneModificationMolecularMutagenesisNeurodegenerative DisordersPharmaceutical PreparationsPharmacologic SubstancePharmacologyPhasePhospholipid InteractionPhospholipidsPhotoaffinity LabelsPlayPositioning AttributeProteinsReagentResearchResearch PersonnelResearch Project GrantsRoleSiteSpecificitySterolsStructural ModelsStructureSynaptic TransmissionSystemTailTechniquesTestingWorkaddictionanalogbiophysical techniquesdesensitizationdesignfield studyion mobilitymethanethiosulfonatemutantneurosteroidsnovelpatch clampreceptorreconstitutionsmall moleculesmall molecule therapeuticsstoichiometry
项目摘要
PROJECY SUMMARY/ ABSTRACT
Pentameric ligand-gated ion channels (pLGICs) play a primary role in synaptic transmission, and are modulated
by a variety of endogenous molecules, including phospholipids, sterols, and fatty acids. pLGICs are also
modulated by small molecule therapeutics (e.g. anesthetics and anti-epileptics). The structural mechanism by
which phospholipids modulate pLGICs is poorly understood. Anionic phospholipids are allosteric modulators of
mammalian pLGICs, and structural studies suggest that phospholipid binding sites overlaps with binding sites of
small molecules such as neuroteroids. The goal of this project is to investigate the hypothesis that lipids and
certain allosteric modulating drugs bind to specific sites on pLGICs, and that these drugs induce their modulatory
effect through a positive, or negative, effect on lipid binding. To accomplish this goal, I will use a combination of
cutting edge techniques, including native mass spectrometry (MS), covalent chemical modification, and patch-
clamp recordings of giant liposomes of defined lipid composition. To apply these techniques, I will use the
prototypical prokaryotic pLGIC, Erwinia ligand-gated ion channel (ELIC), as a tractable model system. ELIC is
an ideal system for MS and readily permits expression and purification of mutant proteins for biochemical and
reconstitution studies. Work in the Cheng lab has measured direct binding of phospholipids to ELIC by MS, and
demonstrated that specific binding of anionic phospholipids reduces desensitization in ELIC. Building upon this
work, this research project will address two aims. The first is to determine the specificity and sites of phospholipid
binding that mediate their modulatory effects on ELIC. I hypothesize that phospholipid head group charge
determines the lipid binding affinity to ELIC, but that the structure of the hydrophobic tail (e.g. length and position
of unsaturations) is the critical determinant of the native modulatory effect. Phospholipid binding affinity and
stoichiometry will be determined by MS. The functionally-relevant binding sites for phospholipids will be
determined using mutagenesis and chemical modification with methanethiosulfonate (MTS) reagents. The second
aim is to elucidate the interaction between phospholipids and allosterically modulating drugs in relation to ELIC
binding and modulation. Within this aim I will determine the sites of binding of allopregnanalone (alloP) in ELIC
using photo-affinity labels, and then examine the effect this labeling (or non-covalent binding in MS) has on
phospholipid binding. Preliminary results indicate that alloP enhances ELIC desensitization, and I hypothesize
that alloP induces its pharmacologic effect by competing for binding of sites otherwise occupied by
phospholipids. Functional studies in liposomes will determine whether alloP competitively or non-competitively
antagonize anionic phospholipid effect. This work will be foundational in understanding the modulation of
pLGICs by relevant small molecules. The experimental framework developed within this proposal will be critical
in understanding the mechanism of channel modulation by other bioactive lipids and small molecule modulators.
项目摘要/摘要
项目成果
期刊论文数量(0)
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科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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John T Petroff 其他文献
John T Petroff 的其他文献
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{{ truncateString('John T Petroff ', 18)}}的其他基金
Structural determinants of lipid modulation of ligand-gated ion channels
配体门控离子通道脂质调节的结构决定因素
- 批准号:
10471051 - 财政年份:2020
- 资助金额:
$ 6.64万 - 项目类别:
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