P2X receptors are trimers, in which each subunit has intracellular N and C termini, two membrane-spanning domains, and a large ectodomain (North, 2002; Kawate et al. 2009). The ATP binding sites are located at the interface between two subunits, and several key residues for agonist docking have been identified. These include Lys69 in the P2X2 receptor, which is suggested to bind to oxygen atoms on the gamma phosphate of ATP. In the homotrimeric receptor, substitution by alanine at this position abolishes the response to ATP (Jiang et al., 2000). We constructed eight concatenated cDNAs, each of which encoded three joined P2X2 subunits, where the mutation K69A was introduced into none, one, two or three of these subunits. All had similar plasma membrane expression when expressed in human embryonic kidney cells, as detected by biotinylation and Western blotting using an epitope tag present in each subunit. There was no evidence of significant degradation of the concatemers, except that a very faint band was sometimes observed corresponding in molecular weight to a dimer. We recorded membrane currents evoked by ATP. Cells expressing the concatemer with Lys at position 69 in each of the three subunits (KKK) had properties comparable to the wild type receptor, although maximal currents were about 50% of those observed with expression of single subunits. Concatemers that contained Lys69 in two of the three domains gave robust currents in response to ATP (KKA=KAK>AKK). Maximal currents were about 50% of those observed with KKK, but the effective concentrations of ATP were in the range of those required to activate receptors formed by expression of monomeric subunits. The activation of the currents (using concentrations of ATP that gave half-maximal current) was slower when concatemers contained one K69A mutation. Outside-out patch recording showed that these constructs had unitary conductances (19 – 23 pS) not different from wild type P2X2 receptors formed from monomer expression, or the KKK construct. Concatemers with only one Lys-containing subunit also showed very small currents in two cases (KAA, AAK) and no current in the other (AKA). We can not exclude the possibility that these result from small amounts of degradation of the concatenated construct. Concatemers in which all three Lys residues at position 69 were replaced by Ala (AAA) did not respond to ATP. The results suggest that ATP can activate P2X receptors which have only two of the three binding sites intact, and that these channels open to a unitary conductance that is not different from that observed in wild type channels.