Gating and conduction of ATP-gated ion channels

P2X受体是由细胞外ATP激活的递质门控离子通道。分布、拓扑、 该家族七个成员(P2X1.7)的药理和生理学都有很好的文献记载。相比之下， 对信号转导途径知之甚少。我们假设受体的激活涉及以下几个方面 步骤：首先，ATP结合到蛋白质复合体的胞外表面的一个位置。第二，占用这块土地 导致允许离子传导发生的通道孔形状的改变。第三，钠和钙流动 顺着它们的电化学梯度进入细胞。第四，Na*的内流使细胞过度兴奋 去极化膜和Caz*的内流触发许多细胞特异性的Sequella，如肌肉 收缩、神经递质释放和感觉。在某些受体亚型中存在一个额外的FIRH步骤 (P2X2，4._)当施加ATP超过几秒钟时；在这里，毛孔最窄的部分膨胀到 允许较大的阳离子，如N-甲基-D-葡萄糖胺(NMDG)和阳离子菁染料？O-PRO-1，渗透到 频道。扩张的功能序列包括起泡、微泡形成和细胞死亡，这些动作可能涉及 受体细胞内C末端尾巴的分子内和/或分子间相互作用。这些实验的目的是 本提案中概述的目的是更好地描述在门控、传导、 和毛孔扩张。在第一个目标中，我们使用几种技术通过均相和非均相来量化离子通量。 我们将这些通量与递质门控离子通道的其他成员中看到的那些通量进行了比较 超级大家庭。此外，我们使用定点突变来确定毛孔内调节通透性的区域。 和穿过表面膜的通量。在接下来的两个目标中，我们将研究通道的分子运动 门控和扩张使用两种不同的技术。在第一组实验中，一系列半胱氨酸取代 突变体和具有硫醇活性的二苯甲酮将被用来绘制跨膜内残基的位置。 在应用ATP之前、期间和之后的分段。在第二组实验中，荧光共振 能量转移(FRET)将被用来确定分子内和分子间的距离 三磷酸腺苷。

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 Caz* triggers numerous cell-specific sequella such as muscle contraction, neurotransmitter release, and sensation. An additional fiRh step occurs in some receptor subtypes (P2X2,4._)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, ¿O-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.