Functional and structural identification of amino acid residues of the P2X2 receptor channel critical for the voltage- and [ATP]-dependent gating
Functional and structural identification of amino acid residues of the P2X2 receptor channel critical for the voltage- and [ATP]-dependent gating
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DOI：
10.1113/jphysiol.2009.182824
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发表时间：
2009-12-15
影响因子：
5.5
通讯作者：
Kubo, Yoshihiro
中科院分区：
文献类型：
--
作者：
Keceli, Batu;Kubo, Yoshihiro
已知细胞外ATP门控阳离子通道P2 X（2）显示电压依赖性门控，尽管缺乏经典电压传感器结构域。我们以前观察到，在ATP存在下，超极化诱发的P2 X（2）激活在稳态下取决于[ATP]。随着[ATP]的增加，电导-电压（G-V）关系向更高的去极化电位移动，激活动力学加快。使用由ATP结合步骤和限速门控步骤组成的三态模型，我们成功地再现了电压依赖性门控，包括其[ATP]依赖性。在这项研究中，为了确定电压和ATP依赖性的结构基础，我们分析了各种突变体在非洲爪蟾卵母细胞表达系统下的双电极电压钳。在ATP结合区突变体K308 R中，G-V关系向更超极化的电位转变，没有明显的[ATP]依赖性，并且激活速度比野生型（WT）更快。除了ATP结合步骤的变化之外，还可以通过假设门控步骤的解离速率增加来模拟这些结果。在第一跨膜区（TM 1）有F44 C突变或在TM 2有T339 S突变时，低[ATP]时激活缓慢，而高[ATP]时在所有膜电位下通道都是组成性激活的。这些结果可以通过降低门控步骤的关闭速率来模拟。双突变体K308 R/F44 C和K308 R/T339 S的表型与WT相似，表明TM和ATP结合区突变体抵消了彼此的作用。对其他两种激动剂ADP和AP（4）A对WT的影响的分析表明，静电电荷不是唯一的关键因素。将这些结果与最近报道的晶体结构一起考虑，有人建议，在ATP结合时，被占据的结合位点间接地与TM区域的细胞外末端相互作用，以电压依赖性方式触发门控的构象变化。
The extracellular ATP-gated cation channel P2X(2) is known to show voltage-dependent gating in spite of the absence of a canonical voltage sensor domain. We previously observed that the hyperpolarization-evoked activation of P2X(2) at the steady state in the presence of ATP varied depending on [ATP]. With increasing [ATP], the conductance-voltage (G-V) relationship shifted to more depolarized potentials and the activation kinetics were accelerated. Using a three-state model consisting of an ATP binding step and a rate limiting gating step, we successfully reproduced the voltage-dependent gating including its [ATP] dependence. In this study, in order to identify the structural basis of voltage and ATP dependence, we analysed various mutants in the Xenopus oocyte expression system under two-electrode voltage clamp. In the ATP binding region mutant K308R, the G-V relationship was shifted towards more hyperpolarized potentials, there was no clear [ATP] dependence, and activation was faster than in wild-type (WT). These results could be simulated by assuming an increase in the off rate of the gating step, in addition to changes in the ATP binding step. With F44C mutation in the 1st transmembrane (TM) region (TM1) or T339S in TM2, activation in low [ATP] was slow and the channel was constitutively active at all membrane potentials in high [ATP]. These results could be simulated by reducing the off rate of the gating step. Phenotypes of the double mutants, K308R/F44C and K308R/T339S, were similar to WT, suggesting that TM and ATP binding region mutants offset the effect of each other. Analysis of the effects on WT of two other agonists, ADP and AP(4)A, revealed that the electrostatic charge is not the sole critical factor. Taking these results together with the recently reported crystal structure, it was suggested that upon binding of ATP, the occupied binding site indirectly interacts with the extracellular end of the TM regions to trigger conformational changes for gating in a voltage-dependent manner.