Gating and conduction of ATP-gated ion channels

描述（由申请人提供）：P2 X受体是由细胞外ATP激活的递质门控离子通道。该家族的七个成员（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，对信号转导途径的了解很少。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上的位点。第二，占据该位点导致通道孔的形状发生变化，从而允许发生离子传导。第三，Na+和Ca 2+沿其电化学梯度向下流动并进入电池。第四，Na+的向内流动通过使膜去极化而使细胞过度兴奋，并且Ca 2+的向内流动触发许多细胞特异性后遗症，例如肌肉收缩、神经递质释放和感觉。当ATP作用超过几秒钟时，在某些受体亚型（P2 X2，4.7）中会发生额外的第五步;此时，孔的最西边部分扩张到允许较大的阳离子（如N-甲基-D-葡糖胺（NMDG）和阳离子花青染料YO-PRO-1）渗透通道的尺寸。扩张的功能性后遗症包括起泡、微泡形成和细胞死亡，这些作用可能涉及受体的细胞内C末端尾的分子内和/或分子间相互作用。本提案中概述的实验的目标是提供门控、传导和孔扩张期间P2 X通道的动力学的更好描述。在第一个目标中，我们使用几种技术来量化离子通量通过同聚体和异聚体P2 X受体，我们比较这些通量中看到的其他成员的发射门控离子通道超家族。此外，我们使用定点诱变来确定孔内的结构域，调节渗透性和跨表面膜的通量。在接下来的两个目标中，我们使用两种不同的技术来研究通道在门控和膨胀过程中的分子运动。在第一组实验中，一系列半胱氨酸取代的突变体和巯基反应性二苯甲酮将用于绘制ATP应用之前、期间和之后跨膜片段内残基的位置。在第二组实验中，将使用荧光共振能量转移（FRET）来确定在不存在和存在ATP的情况下的分子内和分子间距离。

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.