Allosteric nature of P2X receptor activation probed by photoaffinity labelling
Allosteric nature of P2X receptor activation probed by photoaffinity labelling
复制标题
DOI：
10.1111/j.1476-5381.2012.02083.x
复制
发表时间：
2012-11-01
影响因子：
7.3
通讯作者：
Mourot, A.
中科院分区：
文献类型：
--
作者：
Bhargava, Y.;Rettinger, J.;Mourot, A.
背景和目的在P2 X受体中，在相邻亚基之间的界面处的激动剂结合被有效地转换为离子通道门控。然而，结合和门控之间的关系很难研究，因为激动剂不断结合和解除结合。在这里，我们将激动剂共价结合在P2 X受体的结合口袋中，并研究结合位点的占据如何影响通道的门控能力。实验方法我们使用了一种策略，将激动剂拴在它们的ATP结合口袋中，同时使用电生理学探测离子通道门控。激动剂2 '，3'-O-（4-benzoylbenzoyl）-ATP（BzATP）是ATP的光亲和类似物，使我们能够捕获处于不同激动剂结合状态的大鼠同聚体P2 X2受体和P2 X2/1受体嵌合体。UV光用于控制受体的共价占据程度。关键结果P2 X2/1受体嵌合体BzATP复合物的照射导致持续电流，即使在广泛洗脱后也持续存在，这与激动剂BzATP的光化学束缚和受体在开放状态下的捕获一致。用BzATP部分标记引发随后的激动剂结合，并调节完全和部分激动剂的门控效率。结论和意义我们的光标记策略为P2 X受体的激活机制提供了新的分子见解。我们在这里表明，与完整的激动剂分子引发导致随后的激动剂结合后的门控效率的增加。
BACKGROUND AND PURPOSE In P2X receptors, agonist binding at the interface between neighbouring subunits is efficiently transduced to ion channel gating. However, the relationship between binding and gating is difficult to study because agonists continuously bind and unbind. Here, we covalently incorporated agonists in the binding pocket of P2X receptors and examined how binding site occupancy affects the ability of the channel to gate. EXPERIMENTAL APPROACH We used a strategy for tethering agonists to their ATP-binding pocket, while simultaneously probing ion channel gating using electrophysiology. The agonist 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP), a photoaffinity analogue of ATP, enabled us to trap rat homomeric P2X2 receptor and a P2X2/1 receptor chimera in different agonist-bound states. UV light was used to control the degree of covalent occupancy of the receptors. KEY RESULTS Irradiation of the P2X2/1 receptor chimera BzATP complex resulted in a persistent current that lasted even after extensive washout, consistent with photochemical tethering of the agonist BzATP and trapping of the receptors in an open state. Partial labelling with BzATP primed subsequent agonist binding and modulated gating efficiency for both full and partial agonists. CONCLUSIONS AND IMPLICATIONS Our photolabelling strategy provides new molecular insights into the activation mechanism of the P2X receptor. We show here that priming with full agonist molecules leads to an increase in gating efficiency after subsequent agonist binding.