Gating and conduction of ATP-gated ion channels

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。该家族七个成员 (P2X1.7) 的分布、拓扑结构、药理学和生理学都有详细记录。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 与蛋白质复合物细胞外表面的一个位点结合。其次，占据该位置会导致通道孔形状发生变化，从而允许发生离子传导。第三，Na 和 Ca2 沿着电化学梯度流入电池。第四，Na 的向内流动通过使膜去极化而使细胞过度兴奋，而 Ca2 的向内流动引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。当施加 ATP 超过几秒时，某些受体亚型（P2X2，4.7）会发生额外的第五步；在这里，孔的最窄部分扩张到一定尺寸，允许较大的阳离子（如 N-甲基-D-葡萄糖胺 (NMDG) 和阳离子花青染料 YO-PRO-1）渗透到通道中。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细胞内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的目标是更好地描述 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，我们使用多种技术来量化通过同聚和异聚 P2X 受体的离子通量，并将这些通量与递质门控离子通道超家族其他成员中看到的通量进行比较。此外，我们使用定点诱变来识别孔内调节表面膜渗透性和通量的域。在接下来的两个目标中，我们使用两种不同的技术研究通道在门控和扩张过程中的分子运动。在第一组实验中，一系列半胱氨酸取代的突变体和硫醇反应性二苯甲酮将用于在应用 ATP 之前、期间和之后绘制跨膜片段内残基的位置。在第二组实验中，荧光共振能量转移 (FRET) 将用于确定不存在和存在 ATP 时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.