Inter-subunit disulfide locking of the human P2X3 receptor elucidates ectodomain movements associated with channel gating
Inter-subunit disulfide locking of the human P2X3 receptor elucidates ectodomain movements associated with channel gating
复制标题
人 P2X3 受体的亚基间二硫键锁定阐明了与通道门控相关的胞外域运动
DOI：
10.1007/s11302-016-9496-5
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发表时间：
2016
影响因子：
3.5
通讯作者：
Hausmann
中科院分区：
文献类型：
--
作者：
Stephan;Kowalski-Jahn;Schmalzing;Hausmann
P2X3 受体 (P2X3R) 是三聚体 ATP 门控阳离子通道，参与感觉神经传递和炎性疼痛。我们使用 hP2X3R 的同源建模和分子动力学模拟来识别残基的亚基间相互作用，这有助于阐明与 hPX3R 门控相关的构象变化。我们分别鉴定了头部结构域和背鳍结构域的 E112 和 R198 以及相邻亚基下体结构域的 E57 和 T263 之间的离子相互作用，并在 hP3X3R 门控过程中检测到这些结构域的显着重排。亚基间残基对 E112/R198 和 E57/T263 的双突变体循环分析揭示了显着的无相互作用能量。 hP2X3R E112C/R198C 或 E57C/T263C 双半胱氨酸突变体的二硫键锁定显着降低了 ATP 诱导的电流反应。亚基间二硫键交联后电流幅度降低表明，hP2X3R 头部和背鳍结构域或下体结构域水平的二硫键锁定防止了门控诱导的亚基彼此构象重排。 hP2X3R 门控过程中亚基界面的独特重组通常与其他 P2XR 的门控机制一致。取代半胱氨酸的电荷反转诱变和甲硫磺酸盐 (MTS) 修饰表明 E112 和 R198 存在静电相互作用。 E112/R198 相互作用的二硫键锁定和盐桥断裂均降低了 hP2X3R 功能。我们得出结论，头和背鳍结构域的 E112 和 R198 之间的亚基间盐桥分别用于在 hP2X3R 激动剂激活期间控制这些结构域的移动性。
P2X3 receptors (P2X3R) are trimeric ATP-gated cation channels involved in sensory neurotransmission and inflammatory pain. We used homology modeling and molecular dynamic simulations of the hP2X3R to identify inter-subunit interactions of residues that are instrumental to elucidate conformational changes associated with gating of the hPX3R. We identified an ionic interaction between E112 and R198 of the head domain and dorsal fin domain, respectively, and E57 and T263 of the lower body domains of adjacent subunits and detected a marked rearrangement of these domains during gating of the hP3X3R. Double-mutant cycle analysis of the inter-subunit residue pairs E112/R198 and E57/T263 revealed significant interaction-free energies. Disulfide locking of the hP2X3R E112C/R198C or the E57C/T263C double cysteine mutants markedly reduced the ATP-induced current responses. The decreased current amplitude following inter-subunit disulfide cross-linking indicates that disulfide locking of the head and dorsal fin domains or at the level of the lower body domains of the hP2X3R prevents the gating-induced conformational rearrangement of the subunits with respect to each other. The distinct reorganization of the subunit interfaces during gating of the hP2X3R is generally consistent with the gating mechanism of other P2XRs. Charge-reversal mutagenesis and methanethiosulfonate (MTS)-modification of substituted cysteines demonstrated that E112 and R198 interact electrostatically. Both disulfide locking and salt bridge breaking of the E112/R198 interaction reduced the hP2X3R function. We conclude that the inter-subunit salt bridge between E112 and R198 of the head and dorsal fin domains, respectively, serves to control the mobility of these domains during agonist-activation of the hP2X3R.
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影响因子：
56.9
作者：
HIDALGO, P;MACKINNON, R
通讯作者：
MACKINNON, R
核苷酸类似物对 P2X3 受体及其突变体的作用识别激动剂结合袋
影响因子：
3.6
作者：
Riedel T;Wiese S;Leichsenring A;Illes P
通讯作者：
Illes P
影响因子：
4.8
作者：
Bodnar, Mandy;Wang, Haihong;Rubini, Patrizia
通讯作者：
Rubini, Patrizia
影响因子：
64.8
作者：
Althoff, Thorsten;Hibbs, Ryan E.;Banerjee, Surajit;Gouaux, Eric
通讯作者：
Gouaux, Eric
影响因子：
3.5
作者：
Kaczmarek-Hajek, Karina;Loerinczi, Eva;Hausmann, Ralf;Nicke, Annette
通讯作者：
Nicke, Annette