Identification of a distinct desensitisation gate in the ATP-gated P2X2 receptor
Identification of a distinct desensitisation gate in the ATP-gated P2X2 receptor
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DOI：
10.1016/j.bbrc.2019.12.028
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发表时间：
2020-02-26
影响因子：
3.1
通讯作者：
Schmid，Ralf
中科院分区：
文献类型：
--
作者：
Stavrou，Anastasios;Evans，Richard J.;Schmid，Ralf
P2 X受体是三聚体ATP门控离子通道。响应于ATP结合，构象变化导致通道和离子流的打开。在持续的ATP结合过程中，电流会下降，这一过程称为脱敏。脱敏的速率和程度受多种因素影响，例如P2 X2中的T18 A突变使离子通道快速脱敏。我们已经使用这种突变来调查是否门限制离子流是不同的脱敏和关闭状态，通过结合分子建模和半胱氨酸修饰使用MTSET（2-（三甲基铵）乙基甲硫基磺酸盐）。P2 X2受体的同源建模和通道的负空间成像表明限制性门的运动，其中残基T335在脱敏状态下是溶剂可接近的，但在关闭状态下不是。这通过用MTSET探测P2 X2 T18 A/T335 C（快速脱敏）和T335 C（缓慢脱敏）突变体中T335 C的可及性而实验证实，这表明对离子流的屏障在封闭和脱敏状态下是不同的。为了研究T18 A诱导的脱敏开关，我们比较了野生型和T18 A P2 X2受体的分子动力学模拟，这表明脱敏时程的差异是由于T18附近保守残基的氢键网络的结构不稳定。
P2X receptors are trimeric ATP-gated ion channels. In response to ATP binding, conformational changes lead to opening of the channel and ion flow. Current flow can decline during continued ATP binding in a process called desensitisation. The rate and extent of desensitisation is affected by multiple factors, for instance the T18A mutation in P2X2 makes the ion channel fast desensitising. We have used this mutation to investigate whether the gate restricting ion flow is different in the desensitised and the closed state, by combining molecular modelling and cysteine modification using MTSET (2-(Trimethylammonium)ethyl methanethiosulfonate). Homology modelling of the P2X2 receptor and negative space imaging of the channel suggested a movement of the restriction gate with residue T335 being solvent accessible in the desensitised, but not the closed state. This was confirmed experimentally by probing the accessibility of T335C in the P2X2 T18A/T335C (fast desensitisation) and T335C (slow desensitisation) mutants with MTSET which demonstrates that the barrier to ion flow is different in the closed and the desensitised states. To investigate the T18A induced switch in desensitisation we compared molecular dynamics simulations of the wild type and T18A P2X2 receptor which suggest that the differences in time course of desensitisation are due to structural destabilization of a hydrogen bond network of conserved residues in the proximity of T18.