A comparison of the binding characteristics of recombinant P2×1 and P2×2 purinoceptors

Abstract

1 . We have recently provided evidence that [³⁵S]-adenosine 5′-O-[3-thiotriphosphate] ([³⁵S]-ATPγS) can label the human bladder recombinant P_2×1 purinoceptor (human P_2×1 purinoceptor). In this study we have characterized the binding of [³⁵S]-ATPγS to a second P_2X purinoceptor subtype, the rat PC12 phaeochromocytoma cell recombinant P_2×2 purinoceptor (rat P_2×2 purinoceptor), and compared its binding properties with those of both endogenous and recombinant P_2×1 purinoceptors. 2 . Infection of CHO-K1 cells with the rat P_2×2 purinoceptor using Semliki forest virus (SFV) resulted in the expression of high affinity (pK_d = 9.3; B_max = 18.1 pmol mg⁻¹ protein) binding sites for [³⁵S]-ATPγS but not for [³H]-α,β-methylene ATP ([³H]-αβmeATP). Since functional P_2X purinoceptors could be detected electrophysiologically in these cells, but not in non-infected or CHO-K1 cells infected with SFV containing the LacZ gene, these results suggest that the rat P_2×2 purinoceptor can be labelled using [³⁵S]-ATPγS. 3 . The binding characteristics of the rat P_2×2 purinoceptor were compared with those of the human P_2×1 purinoceptor, which was also expressed in the CHO-K1 cells using SFV. A major difference between the two recombinant P_2X purinoceptor types was in the binding characteristics of α,β-methylene ATP (αβmeATP). Thus, in the absence of divalent cations, αβmeATP possessed low affinity for both the human P_2×1 purinoceptor (pIC₅₀ = 7.2) and rat P_2×2 purinoceptor (pIC₅₀ = 7.1) labelled using [³⁵S]-ATPγS. However, when the recombinant P_2X purinoceptors were labelled with [³H]-αβmeATP in the presence of 4 mM CaCl², the affinity of αβmeATP for the human P_2×1 purinoceptor increased (pIC₅₀ for αβmeATP = 8.2), while the affinity of the rat P_2×2 purinoceptor for αβmeATP did not change (pIC₅₀ for αβmeATP = 6.8). 4 . Affinity estimates of 15 other nucleotide analogues for the [³⁵S]-ATPγS binding sites on the two recombinant P_2X purinoceptor subtypes were surprisingly similar (less than 5 fold difference), the only exception being 2′-deoxy ATP which possessed 8 fold higher affinity for rat P_2×2 than for human P_2×1 purinoceptors. In contrast dextran sulphate and the P₂ purinoceptor antagonists, pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid and 4,4′-diisothiocyanatostilbene-2,2′disulphonic acid, possessed 7 to 33 fold higher affinity for the human P_2×1 than for the rat P_2×2 purinoceptor. These data provide a correlation coefficient (r) of 0.894. 5 . There was some evidence for species differences in the P_2×1 purinoceptor. Thus, most nucleotides possessed slightly greater (up to 9–10 fold), while the P₂ purinoceptor antagonists possessed slightly lower (up to 7–16 fold), affinity for the endogenous rat vas deferens and rat bladder P_2×1 purinoceptors than for the human recombinant P_2×1 purinoceptor. These differences were reflected in a slightly lower correlation coefficient, when comparing across species between the human recombinant P_2×1 purinoceptor and the endogenous P_2×1 purinoceptors labelled in either the rat deferens (r = 0.915) or the rat bladder (r = 0.932), than when comparing within species between the endogenous rat vas deferens and rat bladder P_2×1 purinoceptors (r = 0.995). 6 . In summary, [³⁵S]-ATPγS can be used to label the recombinant P_2×1 and P_2×2 purinoceptors. Despite the marked differences reported between these two forms of P_2X purinoceptor in functional studies, the differences in binding studies were more limited. However, a number of antagonists could discriminate between the P_2X purinoceptor subtypes in the binding studies raising expectations that selective antagonists for these receptors can be developed.