The role of positively charged amino acids in ATP recognition by human P2X1 receptors

被引:153
作者
Ennion, S [1 ]
Hagan, S [1 ]
Evans, RJ [1 ]
机构
[1] Univ Leicester, Dept Cell Physiol & Pharmacol, Leicester LE1 9HN, Leics, England
关键词
D O I
10.1074/jbc.M003637200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:29361 / 29367
页数:7
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