Gating and conduction of ATP-gated ion channels

ATP 门控离子通道的门控和传导

• 批准号: 7406271
• 负责人: TERRANCE M EGAN
• 金额: $ 2.17万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7406271
• 关键词: Benzophenones; Binding; Bulla; C-terminal; Cations; Cell Death; Cells; Complex; Cysteine; Esthesia; Family; Family member; Fluorescence Resonance Energy Transfer; Gated Ion Channel; Goals; Ions; Maps; Membrane; Membrane Proteins; Molecular; Motion; Movement; Muscle Contraction; Occupations; P2X-receptor; Permeability; Pharmacology; Physiology; Positioning Attribute; Receptor Activation; Research; Shapes; Signal Transduction Pathway; Site; Site-Directed Mutagenesis; Stretching; Sulfhydryl Compounds; Surface; Tail; Techniques; Testing; benzophenone; cyanine dye; extracellular; member; mutant; neurotransmitter release; receptor; research study; size

P2X receptors are transmitter-gated ion channels activated by extracellular ATP. The distribution, topology, pharmacology, and physiology of the seven members of the family (P2X1.7) are well documented. By contrast, the signal transduction pathway is poorly understood. We hypothesize that activation of the receptor involves the following steps: First, ATP binds to a site on the extracellular surface of the protein complex. Second, occupation of this site results in a change in the shape of the channel pore that permits ion conduction to occur. Third, Na¿ and Ca2¿flow down their electrochemical gradients and into the cell. Fourth, the inward flux of Na* renders the cell hyperexcitable by depolarizing the membrane and the inward flux of Caz* triggers numerous cell-specific sequella such as muscle contraction, neurotransmitter release, and sensation. An additional fiRh step occurs in some receptor subtypes (P2X2,4._)when ATP is applied for more than a few seconds; here, the narrowest part of the pore dilates to a size that allows larger cations like N-methyI-D-glucamine (NMDG) and the cationic cyanine dye, ¿O-PRO-1, to permeate the channel. The functional sequella of dilation include blebbing, microvesiculation, and cell death, actions that may involve intra- and/or inter-molecular interactions of the intracellular C-terminal tail of the receptor. The goal of the experiments outlined in this proposal is to provide a better description of the dynamics of P2X channels during gating, conduction, and pore dilation. In the first aim, we use several techniques to quantify ion flux through homomeric and heteromeric P2X receptors, and we compare these fluxes to those seen in other members of the transmitter-gated ion channel superfamily. Further, we use site-directed mutagenesis to identify domains within the pore that regulate permeability and flux across the surface membrane. In the next two aims, we study the molecular motions of the channel during gating and dilation using two different techniques. In the first set of experiments, an array of cysteine-substituted mutants and thiol-reactive benzophenones will be used to map the position of residues within the transmembrane segments before, during, and after applications of ATP. In the second set of experiments, fluorescence resonance energy transfer (FRET) will be used to determine intra- and inter-molecular distances in the absence and presence of ATP.

P2X受体是由细胞外ATP激活的递质门控离子通道。分布，拓扑，

项目成果

Molecular shape, architecture, and size of P2X4 receptors determined using fluorescence resonance energy transfer and electron microscopy.

使用荧光共振能量转移和电子显微镜测定 P2X4 受体的分子形状、结构和大小。

• DOI: 10.1074/jbc.m804458200
• 发表时间: 2008
• 期刊: The Journal of biological chemistry
• 作者: Young,MarkT;Fisher,JamesA;Fountain,SamuelJ;Ford,RobertC;North,RAlan;Khakh,BaljitS
• 通讯作者: Khakh,BaljitS