Functional characterization of the P2X4 receptor orthologues

The aim of this study was to functionally characterize the recombinant mouse P2X4 receptor and to compare its pharmacological properties with those of the human and rat orthologues. Whole cell recordings were made from rafts of HEK-293 cells stably expressing recombinant mouse, rat or human P2X4 receptors, using Cs-aspartate containing electrodes (3–8 MΩ) in a HEPES-buffered extracellular medium. The agonist potency of ATP at the three species orthologues was similar, with mean EC50 values of 2.3 μM, 1.4 μM and 5.5 μM, respectively. Adenosine-5′-tetraphosphate (AP4) acted as a partial agonist with respect to ATP at the mouse and human P2X4 receptors (EC50=2.6 and 3.0 μM), but was significantly less potent at the rat orthologue (EC50=20.0 μM). α,β-methylene adenosine-5′-triphosphate (α,β-meATP) also acted as a partial agonist, producing 29% of the maximum response at the mouse P2X4 and 24% at the human P2X4 receptor. In contrast to the other species orthologues, α,β-meATP failed to elicit a significant agonist response at rat P2X4 receptors, and was found to act as an antagonist, with an IC50 of 4.6 μM, against 10 μM ATP. Mouse P2X4 receptors were found to be sensitive to the antagonist, pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (IC50=10.5 μM), as were human P2X4 receptors (IC50=9.6 μM). The rat receptor however, showed a low sensitivity to PPADS (IC50>100 μM). All three orthologues were relatively suramin-insensitive (IC50>100 μM) and insensitive to 1-[N,O-Bis(5-isoquinoline sulphonyl)benzyl]-2-(4-phenylpiperazine)ethyl]-5-isoquinoline sulphonamide (KN-62; IC50>3 μM). Our results suggest that the pharmacological properties of the mouse receptor are most similar to the human P2X4 receptor, and differ markedly from the rat receptor. Keywords: Mouse P2X4 receptor, P2X receptor, rundown, AP4, ATP Introduction P2X receptors form a family of ionotropic cation channels, activated by extracellular ATP. To date, cDNAs for seven subunits have been identified (Valera et al., 1994; Brake et al., 1994; Chen et al., 1995; Buell et al., 1996; Bo et al., 1995; Collo et al., 1996; Surprenant et al., 1996) and when expressed in heterologous systems, these produce functional homomeric receptors of unknown stoichiometry. Phenotypic characteristics of purinergic responses in vivo, in some cases, can be explained only by the existence of heteromeric receptors exhibiting novel pharmacological and kinetic phenotypes. Thus, hetero-oligomeric assembly of P2X2/3 (Lewis et al., 1995), P2X1/5 (Torres et al., 1998) and P2X4/6 (Le et al., 1998) subunits have been described in vitro, and the novel pharmacological profiles exhibited by the heteromeric receptors have been shown to be similar to those seen in certain native tissues (e.g. Lewis et al., 1995). Notwithstanding this, the functional phenotypic features of certain homomeric P2X receptor assemblies do resemble phenotypes found in vivo, and thus the study of the operational features of homomeric P2X receptor subunit assemblies remains crucial for our understanding of the role of these channels in physiological function. P2X4 receptors were originally cloned from rat brain (Bo et al., 1995) and were described as being relatively insensitive to the purinergic antagonists, suramin and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS). The agonist, α,β-methylene-ATP (α,β-meATP) was shown to have little effect, and currents elicited by ATP were slowly desensitizing (Buell et al., 1996; Bo et al., 1995). Subsequently, both human (Garcia-Guzman et al., 1997) and murine orthologues of the P2X4 receptor have been isolated (Townsend-Nicholson et al., 1999; Simon et al., 1999), and the limited comparison of the three species orthologues so far performed have revealed operational differences. To date there are no reports describing comparison of the properties of these orthologues in the same study, and even reports characterizing a single orthologue (i.e. the rat) describe some discrepancies. Thus, the response of homomeric rat P2X4 receptors to ATP desensitized slowly (Buell et al., 1996; Collo et al., 1996), although when expressed in oocytes, rat P2X4 receptors required 10–15 min to fully recover from desensitization (Bo et al., 1995). All studies to date show that homomeric rat P2X4 receptors are insensitive to α,β-meATP at concentrations up to 100 μM, when expressed in HEK-293 cells (Buell et al., 1996; Collo et al., 1996), although responses have been obtained from the rat orthologue when expressed in Xenopus oocytes (Le et al., 1998). Bo et al. (1995) originally reported insensitivity to the antagonists, PPADS and suramin; findings supported by a study by Collo et al. (1996). However, studies by Seguela et al. (1996) described modest inhibition by PPADS, and complete blockade by suramin, albeit at high concentrations, while Soto et al. (1996) described modest inhibition by suramin. It should be noted that high concentrations of suramin have been shown to have non-specific effects, and as such cannot be considered as being highly selective for P2 receptors (Balcar, 1995). In contrast to the rat P2X4 receptor, recombinant human P2X4 channels have been shown to be sensitive to PPADS, and slightly sensitive to suramin (Garcia-Guzman et al., 1997; Dhulipala et al., 1998). Allosteric modulation of the human P2X4 receptor by Zn2+ (Soto et al., 1996) and cibacron blue (Miller et al., 1998) has also been demonstrated. The mouse P2X4 receptor is the most recently isolated P2X4 orthologue (Townsend-Nicholson et al., 1999; Simon et al., 1999), has 87 and 94% amino acid identity with the human and rat receptors, respectively, and has been characterized only when expressed in oocytes. Mouse P2X4 subunits produced rapid inward currents in response to ATP, which were potentiated, rather than blocked, by cumulative applications of low concentrations of PPADS or suramin (Townsend-Nicholson et al., 1999). In this study, the whole cell configuration of the patch clamp technique was used to characterize recombinant P2X4 receptors. Each orthologue has been expressed in the same parental cell line, and this represents the first study where a full comparison between species orthologues of P2X4 receptor has been performed under the same experimental conditions. Full characterization of murine P2X4 receptors expressed in a mammalian system has yet to be described, and thus in this study we give particular emphasis to this orthologue. A preliminary account of these studies has been presented to the British Pharmacological Society (Jones et al., 1999).