Conformational flexibility of the agonist binding jaw of the human P2X3 receptor is a prerequisite for channel opening
Conformational flexibility of the agonist binding jaw of the human P2X3 receptor is a prerequisite for channel opening
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DOI：
10.1111/bph.12830
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发表时间：
2014-11-01
影响因子：
7.3
通讯作者：
Riedel，T.
中科院分区：
文献类型：
--
作者：
Kowalski，M.;Hausmann，R.;Riedel，T.
背景和目的假设ATP诱导了配体门控P2X受体结合颚的关闭，最终导致膜通道的打开和阳离子的通量。结合颚的半胱氨酸诱变固定化抑制了ATP诱导的电流反应，但不允许区分结合、门控、亚基组装或转运到质膜的干扰。实验方法：使用疼痛相关人类(h)P2X3受体的分子模型来鉴定氨基酸对，这些氨基酸对位于结合颌骨的唇部，不参与激动剂的结合，但即使在没有ATP的情况下也能强烈地相互接近。关键结果在HEK293细胞或非洲爪蟾细胞中表达的一系列半胱氨酸双突变体hP2X3受体，由于自发形成亚基间二硫键，对α，β -亚甲基ATP （α,β - meATP）表现出抑制的电流反应。用二硫苏糖醇减少这些键逆转了α，β -肉atp跨膜电流的阻断。he -标记的hP2X3受体及其在HEK293 orX中表达的突变体的氨基反应荧光标记。卵母细胞在半胱氨酸双突变体中显示了亚基间交联的形成，此外，证实了它们正确的三聚体组装和细胞表面表达。结论和意义总之，通过在不直接参与激动剂结合的位置取代的半胱氨酸残基的亚基间交联，自发地拧紧hP2X3受体的结合颚，抑制激动剂诱发电流，而不干扰结合、亚基组装或运输。
Background and PurposeIt is assumed that ATP induces closure of the binding jaw of ligand‐gated P2X receptors, which eventually results in the opening of the membrane channel and the flux of cations. Immobilization by cysteine mutagenesis of the binding jaw inhibited ATP‐induced current responses, but did not allow discrimination between disturbances of binding, gating, subunit assembly or trafficking to the plasma membrane.Experimental ApproachA molecular model of the pain‐relevant human (h)P2X3 receptor was used to identify amino acid pairs, which were located at the lips of the binding jaw and did not participate in agonist binding but strongly approached each other even in the absence of ATP.Key ResultsA series of cysteine double mutant hP2X3 receptors, expressed in HEK293 cells orXenopus laevisoocytes, exhibited depressed current responses to α,β‐methylene ATP (α,β‐meATP) due to the formation of spontaneous inter‐subunit disulfide bonds. Reducing these bonds with dithiothreitol reversed the blockade of the α,β‐meATP transmembrane current. Amino‐reactive fluorescence labelling of the His‐tagged hP2X3 receptor and its mutants expressed in HEK293 orX. laevisoocytes demonstrated the formation of inter‐subunit cross links in cysteine double mutants and, in addition, confirmed their correct trimeric assembly and cell surface expression.Conclusions and ImplicationsIn conclusion, spontaneous tightening of the binding jaw of the hP2X3 receptor by inter‐subunit cross‐linking of cysteine residues substituted at positions not directly involved in agonist binding inhibited agonist‐evoked currents without interfering with binding, subunit assembly or trafficking.