Department of Cell Physiology & Pharmacology, University of Leicester, Leicester, UK.
J Neurochem. 2012 Dec;123(5):725-35. doi: 10.1111/jnc.12012. Epub 2012 Oct 11.
Recombinant FlagHis(6) tagged Human P2X1 receptors expressed in HEK293 cells were purified, digested with trypsin and analysed by mass spectroscopy (96% coverage following de-glycosylation and reduction). The receptor was basally phosphorylated at residues S387, S388 and T389 in the carboxyl terminus, a triple alanine mutant of these residues had a modest ~ 25% increase in current amplitude and recovery from desensitization. Chemical modification showed that intracellular lysine residues close to the transmembrane domains and the membrane stabilization motif are accessible to the aqueous environment. The membrane-impermeant cross-linking reagent 3,3'-Dithiobis (sulfosuccinimidylpropionate) (DTSSP) reduced agonist binding and P2X1 receptor currents by > 90%, and modified lysine residues were identified by mass spectroscopy. Mutation to remove reactive lysine residues around the ATP-binding pocket had no effect on inhibtion of agonist evoked currents following DTSSP. However, agonist evoked currents were ~ 10-fold higher than for wild type following DTSSP treatment for mutants K199R, K221R and K199R-K221R. These mutations remove reactive residues distant from the agonist binding pocket that are close enough to cross-link adjacent subunits. These results suggest that conformational change in the P2X1 receptor is required for co-ordination of ATP action.
在 HEK293 细胞中表达的重组 FlagHis(6) 标记的人 P2X1 受体被纯化、用胰蛋白酶消化，并通过质谱分析 (糖基化和还原后 96%的覆盖率)。该受体在羧基末端的残基 S387、S388 和 T389 处被基础磷酸化，这些残基的三丙氨酸突变体的电流幅度增加了约 25%，并且从脱敏中恢复的速度也有所提高。化学修饰表明，靠近跨膜结构域和膜稳定基序的细胞内赖氨酸残基可与水相环境接触。膜不可渗透的交联试剂 3,3'-二硫代双(磺基琥珀酰亚胺基丙酸酯) (DTSSP) 使激动剂结合和 P2X1 受体电流减少了 >90%，并通过质谱鉴定了修饰的赖氨酸残基。去除 ATP 结合口袋周围反应性赖氨酸残基的突变对 DTSSP 后激动剂诱发电流的抑制没有影响。然而，对于 K199R、K221R 和 K199R-K221R 突变体，在 DTSSP 处理后，激动剂诱发的电流比野生型高约 10 倍。这些突变去除了与激动剂结合口袋距离较远但足够接近以交联相邻亚基的反应性残基。这些结果表明，P2X1 受体的构象变化是协调 ATP 作用所必需的。
