The role of positively charged amino acids in ATP recognition by human P2X1 receptors
The role of positively charged amino acids in ATP recognition by human P2X1 receptors
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DOI：
10.1074/jbc.m003637200
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发表时间：
2000-09-22
影响因子：
4.8
通讯作者：
Evans, RJ
中科院分区：
文献类型：
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作者：
Ennion, S;Hagan, S;Evans, RJ
ATP的P2 X受体是配体门控阳离子通道家族。在P2 X受体的胞外环中有11个保守的正电荷。我们已经产生了这些保守残基的点突变体（Lys -> Arg、Lys -> Ala、Arg -> Lys或Arg -> Ale），以确定它们对带负电荷的ATP结合的贡献，ATP诱发浓度依赖性（EC 50类似于0.8 μ M）对非洲爪蟾卵母细胞中表达的野生型（WT）P2 X（1）受体的脱敏反应，苏拉明在浓度响应曲线中产生平行的位移，估计pK（B）为6.7。在位置Lys-53、Lys-190、Lys-215、Lys-325、Arg-202、Arg-305和Arg-314处的氨基酸取代对ATP效力、时程和/或苏拉明敏感性没有影响或仅具有小的变化。在K70 R和R292 K/A突变体中观察到ATP效力的适度变化（分别降低20倍和100倍）。残基K68 A和K309 A的突变使ATP的效力降低了>1400倍，并延长了P2 X（1）受体电流的时程，但对苏拉明拮抗作用没有影响。Lys-68、Lys-70、Arg-292和Lys-309接近受体的预测跨膜结构域，表明ATP结合口袋可能形成于通道前庭附近。
P2X receptors for ATP are a family of ligand-gated cation channels. There are 11 conserved positive charges in the extracellular loop of P2X receptors. We have generated point mutants of these conserved residues (either Lys --> Arg, Lys --> Ala, Arg --> Lys, or Arg --> Ale) in the human P2X(1) receptor to determine their contribution to the binding of negatively charged ATP, ATP evoked concentration-dependent (EC50 similar to 0.8 mu M) desensitizing responses at wild-type (WT) P2X(1) receptors expressed in Xenopus oocytes, Suramin produced a parallel rightward shift in the concentration response curve with an estimated pK(B) of 6.7. Substitution of amino acids at positions Lys-53, Lys-190, Lys-215, Lys-325, Arg-202, Arg-305, and Arg-314 either had no effect or only a small change in ATP potency, time course, and/or suramin sensitivity. Modest changes in ATP potency were observed for mutants at K70R and R292K/A (20- and 100-fold decrease, respectively). Mutations at residues K68A and K309A reduced the potency of ATP by >1400-fold and prolonged the time course of the P2X(1) receptor current but had no effect on suramin antagonism. Lys-68, Lys-70, Arg-292, and Lys-309 are close to the predicted transmembrane domains of the receptor and suggest that the ATP binding pocket may form close to the channel vestibule.