Conductance of P2X4 purinergic receptor is determined by conformational equilibrium in the transmembrane region
Conductance of P2X4 purinergic receptor is determined by conformational equilibrium in the transmembrane region
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DOI：
10.1073/pnas.1600519113
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发表时间：
2016-04-26
影响因子：
11.1
通讯作者：
Shimada, Ichio
中科院分区：
文献类型：
--
作者：
Minato, Yuichi;Suzuki, Shiho;Shimada, Ichio
配体门控离子通道被其配体部分激活，导致电流低于生理性完全激动剂诱发的电流。在P2 X嘌呤能受体的情况下，跨膜区中的阳离子选择性孔在ATP结合至细胞外ATP结合位点时扩大，并且由α，α-亚甲基ATP诱发的电流低于由ATP诱发的电流。然而，P2 X受体部分激活的机制尚不清楚，尽管斑马鱼P2 X（4）受体在apo和ATP结合状态下的晶体结构是可用的。在这里，我们观察到来自M339和M351的NMR信号，它们被引入跨膜区，以及斑马鱼P2 X（4）嘌呤能受体在apo、ATP结合和α，β-亚甲基ATP结合状态下的内源性丙氨酸和甲硫氨酸残基。我们的NMR分析表明，在α，β-亚甲基ATP结合状态下，M339，M351，以及连接ATP结合位点和跨膜区的残基M325和A330，存在于封闭和开放构象之间的构象平衡中，其交换速率比化学位移差慢（< 100 s（-1）），表明在这种状态下，开放构象的小群体导致部分活化。我们的NMR分析还表明，跨膜区在与抑制剂三硝基苯基-ATP结合的状态下采用开放构象，因此拮抗作用是由于离子通路的关闭，除了跨膜区中的孔：即，细胞外区域的横向阳离子通道。
Ligand-gated ion channels are partially activated by their ligands, resulting in currents lower than the currents evoked by the physiological full agonists. In the case of P2X purinergic receptors, a cation-selective pore in the transmembrane region expands upon ATP binding to the extracellular ATP-binding site, and the currents evoked by a, alpha-methylene ATP are lower than the currents evoked by ATP. However, the mechanism underlying the partial activation of the P2X receptors is unknown although the crystal structures of zebrafish P2X(4) receptor in the apo and ATP-bound states are available. Here, we observed the NMR signals from M339 and M351, which were introduced in the transmembrane region, and the endogenous alanine and methionine residues of the zebrafish P2X(4) purinergic receptor in the apo, ATP-bound, and alpha,beta-methylene ATP-bound states. Our NMR analyses revealed that, in the alpha,beta-methylene ATP-bound state, M339, M351, and the residues that connect the ATP-binding site and the transmembrane region, M325 and A330, exist in conformational equilibrium between closed and open conformations, with slower exchange rates than the chemical shift difference (< 100 s(-1)), suggesting that the small population of the open conformation causes the partial activation in this state. Our NMR analyses also revealed that the transmembrane region adopts the open conformation in the state bound to the inhibitor trinitrophenyl-ATP, and thus the antagonism is due to the closure of ion pathways, except for the pore in the transmembrane region: i.e., the lateral cation access in the extracellular region.