Gating and conduction of ATP-gated ion channels

ATP 门控离子通道的门控和传导

• 批准号: 6769685
• 负责人: TERRANCE M EGAN
• 金额: $ 32.2万
• 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 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6769685
• 关键词: adenosine triphosphate; calcium flux; electrophysiology; fluorescence microscopy; fluorescence resonance energy transfer; ionophores; membrane channels; protein quantitation /detection; purinergic receptor; receptor expression; recombinant proteins; site directed mutagenesis; statistics /biometry; tissue /cell culture

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受体是由细胞外ATP激活的递质门控离子通道。该家族的7个成员（P2X1.7）的分布、拓扑结构、药理学和生理学都有很好的记录。相比之下，人们对信号转导途径知之甚少。我们假设受体的激活涉及以下步骤：首先，ATP结合到蛋白质复合物的细胞外表面的一个位点。其次，占据这个位置会导致通道孔的形状发生变化，从而允许离子传导发生。第三，Na+和Ca2+沿着它们的电化学梯度流进细胞。第四，Na+向内的通量通过使膜去极化使细胞过度兴奋，Ca2+向内的通量触发许多细胞特异性的后遗症，如肌肉收缩、神经递质释放和感觉。当ATP作用超过几秒时，在某些受体亚型（P2X2, 4.7）中会发生额外的第五步；在这里，孔的最窄部分扩张到一个大小，允许较大的阳离子，如n -甲基- d -氨基葡萄糖（NMDG）和阳离子花青素染料YO-PRO-1，渗透到通道中。扩张性的功能性后遗症包括起泡、微囊泡和细胞死亡，这些作用可能涉及细胞内受体c端尾部的分子内和/或分子间相互作用。本提案中概述的实验目标是更好地描述P2X通道在门控、传导和孔扩张过程中的动力学。在第一个目标中，我们使用几种技术来量化通过同质和异质P2X受体的离子通量，并将这些通量与透射门控离子通道超家族的其他成员所见的通量进行比较。此外，我们使用位点定向诱变来识别孔内调节表面膜通透性和通量的结构域。在接下来的两个目标中，我们将使用两种不同的技术研究通道在门控和扩张过程中的分子运动。在第一组实验中，将使用一系列半胱氨酸取代突变体和巯基反应性二苯甲酮来绘制ATP应用之前，期间和之后跨膜段内残基的位置。在第二组实验中，荧光共振能量转移（FRET）将被用来确定在没有和存在ATP的情况下分子内和分子间的距离。