Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor
Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor
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DOI：
10.1073/pnas.1118759109
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发表时间：
2012-07-10
影响因子：
11.1
通讯作者：
Nicke, Annette
中科院分区：
文献类型：
--
作者：
Loerinczi, Eva;Bhargava, Yogesh;Nicke, Annette
P2 X受体（P2 XRs）是由细胞外ATP激活的配体门控离子通道。虽然斑马鱼P2 X4 R的晶体结构已经得到解决，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然未知。在这里，我们使用电压钳荧光法研究运动在大鼠P2 X1 R（A118-I125），项目超过拟议的ATP结合位点的富含半胱氨酸的头部域。在用半胱氨酸残基取代时，这些残基中的6个（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-苯甲酰基- ATP时荧光探针的发射显示出显著变化。突变体N120 C和G123 C显示随着电流增加，具有类似动力学的快速荧光降低。相反，突变体P121 C和I125 C表现出缓慢的荧光增加，这似乎与脱敏过程中的电流下降相关。突变体E122 C在ATP和苯甲酰-苯甲酰-ATP的作用下，荧光分别缓慢增强和快速减弱。应用竞争性拮抗剂2 ′，3 ′-O-（2，4，6-trinitrophenyl）-ATP（TNP-ATP）导致N120 C、E122 C和G123 C突变体的荧光发生较大变化，而其他突变体的荧光变化较小或无变化。同样，TNP-ATP诱导的变化在控制突变体远离拟议的ATP结合位点是非常小的或不存在。结合分子模拟研究，我们的数据证实了建议的ATP结合位点，并提供证据表明，ATP在其结合位点的核糖部分面向的解决方案。我们还得出结论，P2 XR激活和脱敏涉及富含半胱氨酸的头部结构域的运动。
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.