Agonist trapped in ATP-binding sites of the P2X2 receptor
Agonist trapped in ATP-binding sites of the P2X2 receptor
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DOI：
10.1073/pnas.1102170108
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发表时间：
2011-05-31
影响因子：
11.1
通讯作者：
Grutter, Thomas
中科院分区：
文献类型：
--
作者：
Jiang, Ruotian;Lemoine, Damien;Grutter, Thomas
atp门控P2X受体是三聚体离子通道，最近被x射线晶体学证实。然而，该结构是在没有ATP的情况下解决的，即使先前被证明对ATP功能很重要的保守氨基酸残基包围的细胞外亚基间空腔被认为可以容纳ATP，但ATP位点的定位仍然难以捉摸。在这里，我们通过邻近依赖的“栓系”反应，在合成的atp衍生的硫醇反应性P2X2激动剂（NCS-ATP）和在P2X2受体的推定结合腔中设计的单个半胱氨酸突变体之间建立共价键，从而定位atp结合位点。通过结合全细胞和单通道记录，我们报告了NCS-ATP在P2X2闭态同源模型中共价和特异性地标记了两个先前未确定的位置N140和L186，它们来自两个相邻的亚基，相隔约18埃，这表明至少存在两种结合模式。在两个位置的栓系反应引发随后的激动剂结合，但具有不同的功能后果。标记一个位置会阻碍随后的ATP功能，从而导致低效率的门控，而另一个位置的系住虽然本身不能产生门控，但会增强随后的ATP功能。因此，我们的研究结果定义了一个大而动态的亚基间atp结合口袋，并表明被困在共价激动剂结合状态的受体在控制离子通道的能力上存在差异。
ATP-gated P2X receptors are trimeric ion channels, as recently confirmed by X-ray crystallography. However, the structure was solved without ATP and even though extracellular intersubunit cavities surrounded by conserved amino acid residues previously shown to be important for ATP function were proposed to house ATP, the localization of the ATP sites remains elusive. Here we localize the ATP-binding sites by creating, through a proximity-dependent "tethering" reaction, covalent bonds between a synthesized ATP-derived thiol-reactive P2X2 agonist (NCS-ATP) and single cysteine mutants engineered in the putative binding cavities of the P2X2 receptor. By combining whole-cell and single-channel recordings, we report that NCS-ATP covalently and specifically labels two previously unidentified positions N140 and L186 from two adjacent subunits separated by about 18 angstrom in a P2X2 closed state homology model, suggesting the existence of at least two binding modes. Tethering reaction at both positions primes subsequent agonist binding, yet with distinct functional consequences. Labeling of one position impedes subsequent ATP function, which results in inefficient gating, whereas tethering of the other position, although failing to produce gating by itself, enhances subsequent ATP function. Our results thus define a large and dynamic intersubunit ATP-binding pocket and suggest that receptors trapped in covalently agonist-bound states differ in their ability to gate the ion channel.