P2X receptors are ligand-gated ion channels gated by extracellular ATP. Several approaches indicate that P2X receptors have a trimeric structure; this is different from other ion channel families, such as glutamate and nicotinic receptors. Whole cell and single channel patch clamp recordings were used to study the functional contribution of each channel subunit in rat P2X₂ receptors expressed in HEK293 cells. We used a previously described reporter mutation [T339K] in the second transmembrane domain of the receptor (Cao et al., 2007). This mutation markedly reduced unitary conductance (7.3 ± 0.4 pS, n = 8, at -120mV), compared to wild type (49.3 ± 4 pS, n = 7, t-test, p < 0.01) channels. Wild type P2X₂ receptors typically show strong inward rectification when activated by ATP (at sub-EC₅₀ concentrations, 3 μM); however, P2X₂[T339K] channels exhibit both inward and outward rectification in whole cell recording. First, we co-expressed wildtype P2X₂ and P2X₂[T339K] subunits in HEK293 cells, and we measured single channel conductance and rectification. After co-expression of wild type and [T33K] subunits, we observed four levels of unitary conductance (I: 8.7 ± 0.1; II: 17.3 ± 1; III: 29.4 ± 1.2; IV: 52.8 ± 1.9 pS, n = 18). The appearance of new levels (II and III) intermediate between those of wild type and [T339K] homomers suggests the assembly of two species of heteromeric channels. Second, we expressed the series of P2X₂ trimeric concatamers that contained the [T339K] substitution in one or more of the first, second or third subunit of the concatamer (i.e. position 1, 2 or 3). Concatamers with three wild type or three [T339K] subunits had unitary conductance similar to those observed with the corresponding monomers (i.e. Thr in position 1, 2 and 3: 45.1 ± 2 pS (n = 9) and with Lys in position 1, 2 and 3: 7.9 ± 0.3 pS, n = 7). Concatamers with one Lys-containing subunit had a unitary conductance similar to level III (in position 1: 26.4 ± 0.8 pS; position 2: 29 ± 1 pS; position 3: 22.9 ± 1.4 pS, n = 4 – 8, ANOVA p > 0.05). Concatamers with two Lys-containing subunits had unitary conductance similar to level II (positions 1+2: 15.4 ± 1.1 pS; positions 1+3: 13.2 ± 0.7 pS; positions 2+3: 13.5 ± 2.3 pS, n = 4 – 8, ANOVA p > 0.05). Thus, the opening levels of the concatamers were independent of the position (i.e. subunit) that carried the mutation [T339K]. Measurement of the rectification shown by concatamers also indicated a progressive contribution by each subunit that was also independent on the position of mutation. In summary, these experiments show that the rat P2X₂ receptors operates as a symmetrical trimer in which each of three subunits contribute equally to the permeation properties of the open channel.