Journal of Biological Chemistry
Volume 275, Issue 38, 22 September 2000, Pages 29361-29367
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MEMBRANE TRANSPORT STRUCTURE FUNCTION AND BIOGENESIS
The Role of Positively Charged Amino Acids in ATP Recognition by Human P2X1 Receptors*

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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 P2X1receptor to determine their contribution to the binding of negatively charged ATP. ATP evoked concentration-dependent (EC50 ∼ 0.8 μm) desensitizing responses at wild-type (WT) P2X1 receptors expressed inXenopus oocytes. Suramin produced a parallel rightward shift in the concentration response curve with an estimated pKB 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 P2X1 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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Published, JBC Papers in Press, May 25, 2000, DOI 10.1074/jbc.M003637200

*

This work was supported by British Heart Foundation Grant PG/98128. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.