Gating and conduction of ATP-gated ion channels

描述(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(P2X1.7)的七个成员的分布、拓扑、药理和生理都有很好的文献记载。相比之下，人们对信号转导途径知之甚少。我们假设该受体的激活包括以下步骤：首先，ATP与蛋白质复合体细胞外表面的一个位置结合。其次，占据这个位置会导致通道孔形状的改变，从而允许离子传导发生。第三，钠和钙沿其电化学梯度流下，进入细胞内。第四，钠的内流通过去极化使细胞过度兴奋，而钙的内流触发了许多细胞特有的序列，如肌肉收缩、神经递质释放和感觉。当应用ATP超过几秒钟时，在一些受体亚型(P2X2，4.7)中发生额外的第五步；在这里，孔的最窄部分膨胀到允许更大的阳离子，如N-甲基-D-葡萄糖胺(NMDG)和阳离子菁染料Yo-PRO-1，渗透到通道中。扩张的功能序列包括起泡、微泡形成和细胞死亡，这些作用可能涉及受体细胞内C末端尾巴的分子内和/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在门控、传导和气孔扩张过程中的P2X通道的动态。在第一个目标中，我们使用几种技术来量化通过同构体和异构体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的其他成员中看到的那些通量进行比较。此外，我们使用定点突变来确定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，我们使用两种不同的技术来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活性的二苯甲酮将被用来绘制在应用ATP之前、期间和之后跨膜片段中残基的位置。在第二组实验中，荧光共振能量转移(FRET)将被用来在没有和存在ATP的情况下确定分子内和分子间的距离。

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.