Gating and conduction of ATP-gated ion channels

ATP 门控离子通道的门控和传导

• 批准号: 7217475
• 负责人: TERRANCE M EGAN
• 金额: $ 30.13万
• 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 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7217475
• 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

DESCRIPTION (provided by applicant): 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 Ca2+ triggers numerous cell-specific sequella such as muscle contraction, neurotransmitter release, and sensation. An additional fifth step occurs in some receptor subtypes (P2X2, 4.7) 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, YO-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.

描述（由申请人提供）：P2X受体是通过细胞外ATP激活的发射机门控离子通道。七个家庭成员的分布，拓扑，药理学和生理学（P2X1.7）已得到充分记录。相比之下，信号转导途径的理解很少。我们假设受体的激活涉及以下步骤：首先，ATP与蛋白质复合物细胞外表面的位点结合。其次，该站点的占用导致通道孔的形状变化，允许离子传导发生。第三，Na+和Ca2+向下流入电化学梯度并进入细胞。第四，Na+的内通量通过去极化CA2+的内向通量来使细胞过度诱发，从而触发许多细胞特异性的后遗症，例如肌肉收缩，神经递质释放和感觉。当ATP施用超过几秒钟时，在某些受体亚型（P2X2，4.7）中发生了额外的第五步。在这里，孔的最狭窄部分扩大到尺寸，该尺寸允许较大的阳离子，例如N-甲基-D-葡糖苷（NMDG）和阳离子氰氰胺染料YO-PRO-1，可以渗透到通道。功能性后遗症的扩张包括流血，微掩膜和细胞死亡，可能涉及受体的细胞内C末端尾巴的内部和/或分子间相互作用。本提案中概述的实验的目的是更好地描述门控，传导和孔隙扩张期间P2X通道的动力学。在第一个目标中，我们使用几种技术来量化通过同源物和异源P2X受体的离子通量，并将这些通量与在发射机门控离子通道超级家族的其他成员中看到的通量进行比较。此外，我们使用位置定向的诱变来识别孔内调节渗透性和通量跨表面膜的域。在接下来的两个目标中，我们使用两种不同的技术研究了通道和扩张过程中通道的分子运动。在第一组实验中，将使用一系列半胱氨酸取代的突变体和硫醇反应性苯苯甲酮来绘制在ATP应用之前，期间和之后，在跨膜段内的残基位置。在第二组实验中，在不存在和存在ATP的情况下，将使用荧光共振能量转移（FRET）来确定分子内和分子间距离。