A comparison of the binding characteristics of recombinant P2×1 and P2×2 purinoceptors
Authors: Michel Anton D. , Lundström Kenneth , Buell Gary N. , Surprenant Annmarie , Valera Soledad , Humphrey Patrick P.A. 
Published: July 2012

. We have recently provided evidence that [35S]‐adenosine 5′‐O‐[3‐thiotriphosphate] ([35S]‐ATPγS) can label the human bladder recombinant P2×1 purinoceptor (human P2×1 purinoceptor). In this study we have characterized the binding of [35S]‐ATPγS to a second P2X purinoceptor subtype, the rat PC12 phaeochromocytoma cell recombinant P2×2 purinoceptor (rat P2×2 purinoceptor), and compared its binding properties with those of both endogenous and recombinant P2×1 purinoceptors.
. Infection of CHO‐K1 cells with the rat P2×2 purinoceptor using Semliki forest virus (SFV) resulted in the expression of high affinity (pKd = 9.3; Bmax = 18.1 pmol mg−1 protein) binding sites for [35S]‐ATPγS but not for [3H]‐α,β‐methylene ATP ([3H]‐αβmeATP). Since functional P2X 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 P2×2 purinoceptor can be labelled using [35S]‐ATPγS.
. The binding characteristics of the rat P2×2 purinoceptor were compared with those of the human P2×1 purinoceptor, which was also expressed in the CHO‐K1 cells using SFV. A major difference between the two recombinant P2X 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 P2×1 purinoceptor (pIC50 = 7.2) and rat P2×2 purinoceptor (pIC50 = 7.1) labelled using [35S]‐ATPγS. However, when the recombinant P2X purinoceptors were labelled with [3H]‐αβmeATP in the presence of 4 mM CaCl2, the affinity of αβmeATP for the human P2×1 purinoceptor increased (pIC50 for αβmeATP = 8.2), while the affinity of the rat P2×2 purinoceptor for αβmeATP did not change (pIC50 for αβmeATP = 6.8).
. Affinity estimates of 15 other nucleotide analogues for the [35S]‐ATPγS binding sites on the two recombinant P2X 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 P2×2 than for human P2×1 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 P2×1 than for the rat P2×2 purinoceptor. These data provide a correlation coefficient (r) of 0.894.
. There was some evidence for species differences in the P2×1 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 P2×1 purinoceptors than for the human recombinant P2×1 purinoceptor. These differences were reflected in a slightly lower correlation coefficient, when comparing across species between the human recombinant P2×1 purinoceptor and the endogenous P2×1 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 P2×1 purinoceptors (r = 0.995).
. In summary, [35S]‐ATPγS can be used to label the recombinant P2×1 and P2×2 purinoceptors. Despite the marked differences reported between these two forms of P2X purinoceptor in functional studies, the differences in binding studies were more limited. However, a number of antagonists could discriminate between the P2X purinoceptor subtypes in the binding studies raising expectations that selective antagonists for these receptors can be developed.