Voltage- and [ATP]-dependent gating of the P2X(2) ATP receptor channel.
Voltage- and [ATP]-dependent gating of the P2X(2) ATP receptor channel.
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DOI：
10.1085/jgp.200810002
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发表时间：
2009-01
期刊：
影响因子：
--
通讯作者：
Kubo Y
中科院分区：
文献类型：
--
作者：
Fujiwara Y;Keceli B;Nakajo K;Kubo Y
P2X受体是由细胞外三磷酸腺苷(ATP)激活的配体门控阳离子通道。然而，在施加ATP后的稳态期间观察到的P2X2通道电流表现出电压依赖性；在超极化时，内向电流逐渐增加。我们利用非洲爪哇卵母细胞表达系统和双电极电压钳对这一“激活”阶段进行了定量分析。我们表征了在不同[ATP]存在下的电导-电压关系，并观察到随着[ATP]的增加，电导-电压关系向更去极化的电位方向移动。通过分析通道从闭合状态到开放状态转换的速率常数，我们证明了P2X2的门控是以一种复杂的方式决定的，包括膜电压和ATP结合。在表达P2X2的HEK293细胞中，即使在密集灌流后，即使用内向外膜片钳也同样记录到活化期，排除了门控是由于内源性卵子阻滞剂(S)阻断/解除阻断的可能性。我们通过在第二个跨膜(TM)螺旋上替换甘氨酸残基(G344)来研究它的结构基础，这可能提供了一个可能介导“门控”的扭结。我们发现，通过G344A突变体的内向电流在超极化时瞬间增加，而不是逐渐增加，而G344P突变体保留了一个比野生型(WT)慢的激活期。在G344A的背景下进行甘氨酸扫描诱变，我们可以通过在第二TM的中间引入甘氨酸残基来恢复激活期。这些结果表明，G344的灵活性有助于实现电压依赖门控。最后，我们假设了一个由一个快速的ATP结合步骤和一个随后的门控步骤组成的三态模型，并估计了后者在P2X2-WT中的速率常数。然后，我们使用计算的速率常数进行了模拟分析，并成功地复制了实验观察到的结果，即随着[ATP]的增加而加速的电压依赖的激活。
P2X receptors are ligand-gated cation channels activated by extracellular adenosine triphosphate (ATP). Nonetheless, P2X2 channel currents observed during the steady-state after ATP application are known to exhibit voltage dependence; there is a gradual increase in the inward current upon hyperpolarization. We used a Xenopus oocyte expression system and two-electrode voltage clamp to analyze this “activation” phase quantitatively. We characterized the conductance–voltage relationship in the presence of various [ATP], and observed that it shifted toward more depolarized potentials with increases in [ATP]. By analyzing the rate constants for the channel's transition between a closed and an open state, we showed that the gating of P2X2 is determined in a complex way that involves both membrane voltage and ATP binding. The activation phase was similarly recorded in HEK293 cells expressing P2X2 even by inside-out patch clamp after intensive perfusion, excluding a possibility that the gating is due to block/unblock by endogenous blocker(s) of oocytes. We investigated its structural basis by substituting a glycine residue (G344) in the second transmembrane (TM) helix, which may provide a kink that could mediate “gating.” We found that, instead of a gradual increase, the inward current through the G344A mutant increased instantaneously upon hyperpolarization, whereas a G344P mutant retained an activation phase that was slower than the wild type (WT). Using glycine-scanning mutagenesis in the background of G344A, we could recover the activation phase by introducing a glycine residue into the middle of second TM. These results demonstrate that the flexibility of G344 contributes to the voltage-dependent gating. Finally, we assumed a three-state model consisting of a fast ATP-binding step and a following gating step and estimated the rate constants for the latter in P2X2-WT. We then executed simulation analyses using the calculated rate constants and successfully reproduced the results observed experimentally, voltage-dependent activation that is accelerated by increases in [ATP].
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影响因子：
56.9
作者：
Doyle, DA;Cabral, JM;MacKinnon, R
通讯作者：
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