Pratt Emily B, Brink Thaddeus S, Bergson Pamela, Voigt Mark M, Cook Sean P
Oregon Health and Science University L-106, Portland, Oregon 97201-3098, USA.
J Neurosci. 2005 Aug 10;25(32):7359-65. doi: 10.1523/JNEUROSCI.5189-04.2005.
P2X3 receptors desensitize within 100 ms of channel activation, yet recovery from desensitization requires several minutes. The molecular basis for this slow rate of recovery is unknown. We designed experiments to test the hypothesis that this slow recovery is attributable to the high affinity (< 1 nM) of desensitized P2X3 receptors for agonist. We found that agonist binding to the desensitized state provided a mechanism for potent inhibition of P2X3 current. Sustained applications of 0.5 nM ATP inhibited > 50% of current to repetitive applications of P2X3 agonist. Inhibition occurred at 1000-fold lower agonist concentrations than required for channel activation and showed strong use dependence. No inhibition occurred without previous activation and desensitization. Our data are consistent with a model whereby inhibition of P2X3 by nanomolar [agonist] occurs by the rebinding of agonist to desensitized channels before recovery from desensitization. For several ATP analogs, the concentration required to inhibit P2X3 current inversely correlated with the rate of recovery from desensitization. This indicates that the affinity of the desensitized state and recovery rate primarily depend on the rate of agonist unbinding. Consistent with this hypothesis, unbinding of [32P]ATP from desensitized P2X3 receptors mirrored the rate of recovery from desensitization. As expected, disruption of agonist binding by site-directed mutagenesis increased the IC50 for inhibition and increased the rate of recovery.
P2X3受体在通道激活后100毫秒内会发生脱敏，但从脱敏状态恢复却需要几分钟。这种缓慢恢复速率的分子基础尚不清楚。我们设计了实验来检验这一假说，即这种缓慢恢复是由于脱敏的P2X3受体对激动剂具有高亲和力（<1 nM）。我们发现激动剂与脱敏状态的结合提供了一种有效抑制P2X3电流的机制。持续应用0.5 nM ATP可抑制重复应用P2X3激动剂时>50%的电流。抑制发生时的激动剂浓度比通道激活所需浓度低1000倍，且表现出强烈的使用依赖性。没有先前的激活和脱敏就不会发生抑制。我们的数据与一种模型相符，即纳摩尔浓度的[激动剂]对P2X3的抑制是通过激动剂在从脱敏状态恢复之前重新结合到脱敏通道上实现的。对于几种ATP类似物，抑制P2X3电流所需的浓度与从脱敏状态恢复的速率呈负相关。这表明脱敏状态的亲和力和恢复速率主要取决于激动剂解离的速率。与这一假说一致，[32P]ATP从脱敏的P2X3受体上的解离反映了从脱敏状态恢复的速率。正如预期的那样，通过定点诱变破坏激动剂结合会增加抑制的IC50并提高恢复速率。
