Voltage- and ATP-dependent structural rearrangements of the P2X2 receptor associated with the gating of the pore
Voltage- and ATP-dependent structural rearrangements of the P2X2 receptor associated with the gating of the pore
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DOI：
10.1113/jphysiol.2014.278507
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发表时间：
2014-11-01
影响因子：
5.5
通讯作者：
Kubo，Yoshihiro
中科院分区：
文献类型：
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作者：
Keceli，Batu;Kubo，Yoshihiro
关键点我们以前报道过ATP受体通道P2 X2显示门控，它不仅依赖于ATP，而且依赖于膜电压，本研究的目的是捕获与电压和ATP依赖性门控相关的P2 X2的结构重排。ATP结合位点和通道孔。我们用两电极电压钳记录电流，分析了Cd 2+对半胱氨酸残基的修饰率，发现Cd 2+对315和67位的半胱氨酸残基进行桥连，导致电流下降，超极化时修饰速度比去极化时快，并且在ATP存在下比在不存在下更快。结果表明，P2 X2连接子区域的结构重排与电压相关，P2 X2是一种细胞外ATP门控阳离子通道，尽管缺乏经典的电压传感器，但它具有电压依赖性门控特性。它是一种三聚体，其中每个亚基具有两个跨膜螺旋和一个大的胞外结构域。三个亚基间ATP结合位点通过β链连接到孔形成跨膜（TM）结构域。我们分析了ATP结合位点和TM结构域之间的连接链在配体结合和电压变化时的结构重排，电生理学在异种卵母细胞中，使用携带工程巯基修饰的半胱氨酸残基的突变体。(1)我们证明了双突变体D315 C和I67 C（分别在β-14和β-1处）在用还原剂二硫苏糖醇（DTT）处理后显示电流振幅增加2至4倍。硫醇反应性金属Cd 2+的应用由于D315 C和I67 C之间的键形成而引起电流下降。在野生型（WT）或单点突变体中未观察到这种效应。(2)在超极化和去极化条件下，采用不同的脉冲方案，以及在存在和不存在ATP的情况下，分析了Cd 2+诱导的电流下降。(3)在存在ATP的情况下可以清楚地观察到由Cd 2+诱导的电流下降，但在不存在ATP的情况下不清楚，显示出状态依赖性修饰。(4)在ATP的存在下，超极化条件下的Cd 2+修饰明显快于去极化条件，显示出连接链的电压依赖性结构重排。(5)使用串联三聚体构建体（TTC）进行的实验表明，Cd 2+在315和67之间的桥接不是亚基内，而是亚基间。(6)最后，我们对孔突变体T339 S进行了类似的分析，这使得通道激活电压不敏感。T339 S在超极化和去极化条件下的Cd ~（2+）修饰率相似。将这些结果结合在一起，我们证明了P2 X2受体通道的接头区域的结构重排不仅由配体结合诱导，而且由膜电位变化诱导。
Key pointsWe previously reported that the ATP receptor channel P2X2 shows gating which depends not only on ATP but also on membrane voltage, in spite of the absence of a canonical voltage sensor.The purpose of the present study was to capture the structural rearrangements of P2X2 associated with voltage‐ and ATP‐dependent gating.Cysteine residues were introduced by mutation at Asp315 and Ile67 in the linker region located between the ATP binding site and the channel pore. We analysed the modification rate of cysteine residues by Cd2+by recording current under two‐electrode voltage clamp usingXenopusoocytes.Two cysteine residues at 315 and 67 were bridged by Cd2+resulting in current decline, and the speed of modification was faster at hyperpolarized than at depolarized potential, and also faster in the presence than in the absence of ATP.The bridging by Cd2+between 315 and 67 was not intra‐ but inter‐subunit.The results demonstrate the structural rearrangements in the linker region of P2X2 associated with voltage‐ and ATP‐dependent gating of the pore.AbstractP2X2 is an extracellular ATP‐gated cation channel which has a voltage‐dependent gating property even though it lacks a canonical voltage sensor. It is a trimer in which each subunit has two transmembrane helices and a large extracellular domain. The three inter‐subunit ATP binding sites are linked to the pore forming transmembrane (TM) domains by β‐strands. We analysed structural rearrangements of the linker strands between the ATP binding site and TM domains upon ligand binding and voltage change, electrophysiologically inXenopusoocytes, using mutants carrying engineered thiol‐modifiable cysteine residues. (1) We demonstrated that the double mutant D315C&I67C (at β‐14 and β‐1, respectively) shows a 2‐ to 4‐fold increase in current amplitude after treatment with a reducing reagent, dithiothreitol (DTT). Application of the thiol‐reactive metal Cd2+induced current decline due to bond formation between D315C and I67C. This effect was not observed in wild type (WT) or in single point mutants. (2) Cd2+‐induced current decline was analysed in hyperpolarized and depolarized conditions with different pulse protocols, and also in the presence and absence of ATP. (3) Current decline induced by Cd2+could be clearly observed in the presence of ATP, but was not clear in the absence of ATP, showing a state‐dependent modification. (4) In the presence of ATP, Cd2+modification was significantly faster in hyperpolarized than in depolarized conditions, showing voltage‐dependent structural rearrangements of the linker strands. (5) Experiments using tandem trimeric constructs (TTCs) with controlled number and position of mutations in the trimer showed that the bridging by Cd2+between 315 and 67 was not intra‐ but inter‐subunit. (6) Finally, we performed similar analyses of a pore mutant T339S, which makes the channel activation voltage insensitive. Cd2+modification rates of T339S were similar in hyperpolarized and depolarized conditions. Taking these results together, we demonstrated that structural rearrangements of the linker region of the P2X2 receptor channel are induced not only by ligand binding but also by membrane potential change.