Ishchenko Yevheniia, Novosolova Nataliia, Khafizov Kamil, Bart Geneviève, Timonina Arina, Fayuk Dmitriy, Skorinkin Andrei, Giniatullin Rashid
A. I. Virtanen Institute, University of Eastern Finland , Kuopio, Finland.
Moscow Institute of Physics and Technology , Dolgoprudny, Moscow Region, Russian Federation.
Biochemistry. 2017 Jul 5;56(26):3394-3402. doi: 10.1021/acs.biochem.7b00258. Epub 2017 Jun 26.
Serine 275, a conserved residue of the left flipper region of ATP-gated P2X3 receptors, plays a key role in both agonist binding and receptor desensitization. It is conserved in most of the P2X receptors except P2X7 and P2X6. By combining experimental patch-clamp and modeling approaches, we explored the role of the corresponding residue in the rat P2X7 receptor (rP2X7) by replacing the phenylalanine at position 288 with serine and characterizing the membrane currents generated by either the wild-type (WT) or the mutated rP2X7 receptor. F288S, an rP2X7 mutation, slowed the deactivation subsequent to 2 and 20 s applications of 1 mM ATP. F288S also prevented sensitization (a progressive current growth) observed with the WT in response to a 20 s application of 1 mM ATP. Increasing the ATP concentration to 5 mM promoted sensitization also in the mutated rP2X7 receptor, accelerating the deactivation rate to typical WT values. YO-PRO1 uptake in cells expressing either the WT or the F288S P2X7 receptor was consistent with recorded membrane current data. Interestingly, in the human P2X7 (hP2X7) receptor, substitution Y288S did not change the deactivation rate, while the Y288F mutant generated a "rat-like" phenotype with a fast deactivation rate. Our combined experimental, kinetic, and molecular modeling data suggest that the rat F288S novel phenotype is due to a slower rate of ATP binding and/or unbinding and stabilization of nonsensitized receptor states.
丝氨酸275是ATP门控P2X3受体左鳍状结构域的一个保守残基，在激动剂结合和受体脱敏中均起关键作用。除P2X7和P2X6外，它在大多数P2X受体中都保守。通过结合实验性膜片钳和建模方法，我们将大鼠P2X7受体（rP2X7）第288位的苯丙氨酸替换为丝氨酸，并对野生型（WT）或突变型rP2X7受体产生的膜电流进行表征，以探究相应残基在rP2X7受体中的作用。rP2X7突变体F288S在施加1 mM ATP 2秒和20秒后，失活速度减慢。F288S还可阻止WT在施加1 mM ATP 20秒时出现的敏化现象（电流逐渐增大）。将ATP浓度提高到5 mM时，突变型rP2X7受体也会出现敏化现象，失活速率加快至典型的WT值。在表达WT或F288S P2X7受体的细胞中，YO-PRO1摄取与记录的膜电流数据一致。有趣的是，在人P2X7（hP2X7）受体中，Y288S替换并未改变失活速率，而Y288F突变体产生了具有快速失活速率的“大鼠样”表型。我们综合的实验、动力学和分子建模数据表明，大鼠F288S的新表型是由于ATP结合和/或解离速率较慢以及非敏化受体状态的稳定所致。
