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P2X receptors are ATP-gated nonselective cation channels. Functional receptors are assembled as homotrimers or heterotrimers of seven cloned subunits. Each subunit contains two transmembrane domains linked by a large extracellular loop that is required for agonist binding. So far, there is no direct evidence indicating whether the agonist binding site is formed within one subunit or at the interface of two neighboring subunits. Here we used a disulfide cross-linking approach to identify pairs of residues that are in close proximity within the ATP binding site of the P2X 1 homotrimer. Eight amino acid residues that have previously been shown to be essential for high ATP potency (K68, K70, F185, K190, F291, R292, R305, and K309) were substituted by cysteine residues, and the respective mutant subunits were pairwise expressed in Xenopus laevis oocytes. Nonreducing SDS-PAGE analysis of the purified receptors revealed a spontaneous and specific dimer formation between the K68C and F291C mutants. An almost complete cross-link into trimers was achieved with the K68C/F291C double mutant, consistent with the formation of intersubunit disulfide bridges. In support of this interpretation, twoelectrode voltage-clamp analysis of the K68C/F291C mutations introduced into a nondesensitizing P2X 2-1 chimera showed only small ATP-activated currents that, however, increased ϳ60-fold after extracellular application of the reducing agent dithiothreitol. In addition, we show that a K68C/K309C double mutant is nonfunctional and can be functionally rescued by coexpression with nonmutated subunits. Our data are consistent with loops from neighboring P2X subunits forming the ATP-binding site in P2X receptors.

P2X receptors are ATP-gated cation channels and assembled as homotrimers or heterotrimers from seven cloned subunits. Each subunit contains two transmembrane domains connected by a large extracellular loop. We have previously shown that replacement of two conserved residues, K68 and F291, by cysteine residues leads to disulWde cross-linking between neighbouring P2X 1 subunits. Since mutation of these residues results in a reduced ATP potency and cysteine cross-linking is prevented in the presence of ATP, we suggested an inter-subunit ATP binding site. To investigate whether the proximity of these residues is preserved in other P2X subtypes, we tested for spontaneous cystine formation between the corresponding P2X 2 (K69C, F289C), P2X 3 (K63C, F280C), and P2X 4 (K67C, F294C) mutants upon pairwise expression in Xenopus laevis oocytes. Non-reducing SDS-PAGE analysis of the puriWed receptors revealed a speciWc dimer formation between P2X 2 K69C and P2X 2 F289C mutants. Likewise, co-expression of P2X 1 K68C and P2X 2 F289C, but not P2X 1 F291C and P2X 2 K69C, mutants resulted in dimer formation between the respective subunits. Cross-linked P2X 1/2 heteromers showed strongly reduced or absent function that was selectively recovered upon treatment with DTT. Crosslinking was less eYcient between P2X 3 or P2X 4 mutants but could be enhanced by the short cysteine-reactive crosslinker MTS-2-MTS. These results show that the spatial proximity and/or orientation of residues analogous to positions K68 and F291 in P2X 1 are preserved in P2X 2 receptors and at one of two possible interfaces in heteromeric P2X 1/2 receptors but appears to be redundant for P2X 3 and P2X 4 receptor function.

No abstract