The role of positively charged amino acids in ATP recognition by human P2X(1) receptors.
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 --> Ala) in the human P2X(1) receptor to determine their contribution to the binding of negatively charged ATP. ATP evoked concentration-dependent (EC(50) approximately 0.8 microm) 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.
Ennion
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Department of Cell Physiology, University of Leicester, P. O. Box 138, Leicester LE1 9HN, United Kingdom.
Hagan
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Evans
R J
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D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D006801:Humans; D008969:Molecular Sequence Data; D018048:Receptors, Purinergic P2 / Q000737:chemistry* / Q000378:metabolism; D055672:Static Electricity
D000596:Amino Acids; D018048:Receptors, Purinergic P2; D000255:Adenosine Triphosphate