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J. Biol. Chem., Vol. 279, Issue 10, 9043-9055, March 5, 2004

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ATP Binding at Human P2X₁ Receptors

CONTRIBUTION OF AROMATIC AND BASIC AMINO ACIDS REVEALED USING MUTAGENESIS AND PARTIAL AGONISTS^*

Jonathan A. Roberts and Richard J. Evans

From the Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester LE1 9HN, United Kingdom

P2X receptors comprise a family of ATP-gated ion channels with the basic amino acids Lys-68, Arg-292, and Lys-309 (P2X₁ receptor numbering) contributing to agonist potency. In many ATP-binding proteins aromatic amino acids coordinate the binding of the adenine group. There are 20 conserved aromatic amino acids in the extracellular ligand binding loop of at least 6 of the 7 P2X receptors. We used alanine replacement mutagenesis to determine the effects of individual conserved aromatic residues on the properties of human P2X₁ receptors expressed in Xenopus oocytes. ATP evoked concentration-dependent (EC₅₀ 1 µM) desensitizing currents at wild-type receptors and for the majority of mutants there was no change (10 residues) or a <6-fold decrease in ATP potency (6 mutants). Mutants F195A and W259A failed to form detectable channels at the cell surface. F185A and F291A produced 10- and 160-fold decreases in ATP potency. The partial agonists 2',3'-O-(4-benzoyl)-ATP (BzATP) and P¹,P⁵-di(adenosine 5')-pentaphosphate (Ap₅A) were tested on a range of mutants that decreased ATP potency to determine whether this resulted predominantly from changes in agonist binding or gating of the channel. At K68A and K309A receptors BzATP and Ap₅A had essentially no agonist activity but antagonized, or for R292A potentiated, ATP responses. At F185A receptors BzATP was an antagonist but Ap₅A no longer showed affinity for the receptor. These results suggest that residues Lys-68, Phe-185, Phe-291, Arg-292, and Lys-309 contribute to ligand binding at P2X₁ receptors, with Phe-185 and Phe-291 coordinating the binding of the adenine ring of ATP.

Received for publication, August 13, 2003 , and in revised form, December 16, 2003.

^* This work was supported by the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 44-116-252-3032; Fax: 44-116-252-5045; E-mail: rje6{at}le.ac.uk.

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