Department of Molecular Biology of Neuronal Signals, Max Planck Institute for Experimental Medicine, 37075 Göttingen, Germany.
Proc Natl Acad Sci U S A. 2012 Jul 10;109(28):11396-401. doi: 10.1073/pnas.1118759109. Epub 2012 Jun 27.
P2X receptors (P2XRs) are ligand-gated ion channels activated by extracellular ATP. Although the crystal structure of the zebrafish P2X4R has been solved, the exact mode of ATP binding and the conformational changes governing channel opening and desensitization remain unknown. Here, we used voltage clamp fluorometry to investigate movements in the cysteine-rich head domain of the rat P2X1R (A118-I125) that projects over the proposed ATP binding site. On substitution with cysteine residues, six of these residues (N120-I125) were specifically labeled by tetramethyl-rhodamine-maleimide and showed significant changes in the emission of the fluorescence probe on application of the agonists ATP and benzoyl-benzoyl-ATP. Mutants N120C and G123C showed fast fluorescence decreases with similar kinetics as the current increases. In contrast, mutants P121C and I125C showed slow fluorescence increases that seemed to correlate with the current decline during desensitization. Mutant E122C showed a slow fluorescence increase and fast decrease with ATP and benzoyl-benzoyl-ATP, respectively. Application of the competitive antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) resulted in large fluorescence changes with the N120C, E122C, and G123C mutants and minor or no changes with the other mutants. Likewise, TNP-ATP-induced changes in control mutants distant from the proposed ATP binding site were comparably small or absent. Combined with molecular modeling studies, our data confirm the proposed ATP binding site and provide evidence that ATP orients in its binding site with the ribose moiety facing the solution. We also conclude that P2XR activation and desensitization involve movements of the cysteine-rich head domain.
P2X 受体（P2XRs）是一种配体门控离子通道，可被细胞外 ATP 激活。虽然已经解析了斑马鱼 P2X4R 的晶体结构，但 ATP 结合的确切模式以及控制通道开放和脱敏的构象变化仍不清楚。在这里，我们使用电压钳荧光法研究了投射到拟议的 ATP 结合位点上的大鼠 P2X1R（A118-I125）的富含半胱氨酸的头部结构域的运动。在用半胱氨酸残基取代时，其中六个残基（N120-I125）被四甲基罗丹明马来酰亚胺特异性标记，并且在应用激动剂 ATP 和苯甲酰苯甲酰-ATP 时，荧光探针的发射显示出显著变化。突变体 N120C 和 G123C 显示出与电流增加相似动力学的快速荧光降低。相比之下，突变体 P121C 和 I125C 显示出缓慢的荧光增加，似乎与脱敏过程中的电流下降相关。突变体 E122C 用 ATP 和苯甲酰苯甲酰-ATP 分别显示出缓慢的荧光增加和快速降低。竞争性拮抗剂 2'，3'-O-（2,4,6-三硝基苯基）-ATP（TNP-ATP）的应用导致 N120C、E122C 和 G123C 突变体的荧光发生大的变化，而其他突变体的荧光变化较小或没有变化。同样，远离拟议的 ATP 结合位点的对照突变体中 TNP-ATP 诱导的变化也很小或不存在。结合分子建模研究，我们的数据证实了拟议的 ATP 结合位点，并提供了证据表明 ATP 在其结合位点中以核糖部分面向溶液的方式定向。我们还得出结论，P2XR 的激活和脱敏涉及富含半胱氨酸的头部结构域的运动。
