Pharmacological characterization of recombinant human and rat P2X receptor subtypes
Introduction
The proposed role of ATP as a functional neurotransmitter and local intercellular signaling molecule (Burnstock, 1972) has gained widespread acceptance since the cloning of cell-surface receptors specifically activated by purine and pyrimidine nucleotides (P2 receptors) (Burnstock, 1996; Williams and Burnstock, 1997). These receptors are sub-classified into two broad groups, P2X and P2Y, based on their structural and functional similarities (Burnstock and Kennedy, 1985; Fredholm et al., 1997). P2X receptors are multimeric ligand-gated ionotropic channels that exhibit a non-selective cation permeability, and share a common structural motif characterized by two transmembrane spanning domains connected by an extracellular loop. P2Y receptors are members of the G protein-coupled receptor family (metabotropic receptors), characterized by seven transmembrane spanning domains and a signaling mechanism dependent on heterotrimeric G proteins (Harden et al., 1995).
To date, seven functional P2X and five functional P2Y receptors have been identified by molecular cloning techniques. P2Y receptors are activated by a variety of nucleotide ligands, including purine and pyrimidine nucleotides both in the diphosphate and triphosphate forms (Harden et al., 1995; Communi and Boeynaems, 1997). In contrast, P2X receptors appear to share ATP as their endogenous ligand and may exhibit signaling specificity based primarily on differential tissue localization, agonist sensitivity, and quaternary structure (i.e., homo- vs. heteromultimerization) (Williams and Burnstock, 1997). In addition to the P2X and P2Y receptor families, additional nucleotide receptors have been postulated on the basis of radioligand binding assays and functional activity. However, these novel receptor subtypes, including P2T (activated by ADP) (Hourani and Hall, 1996) and P2D (activated by diadenosine polyphosphates) (Miras-Portugal et al., 1996) receptors, have not yet been identified at the molecular level.
The recombinant P2X1 receptor has been expressed transiently in Xenopus oocytes and HEK293 cells, where it exhibits sensitivity to the agonist αβ-methylene ATP (αβ-meATP) and rapid desensitization kinetics (Valera et al., 1994). In contrast, the P2X2 receptor, originally cloned from rat PC12 cells, is insensitive to αβ-meATP and desensitizes slowly after agonist activation (Brake et al., 1994; Evans et al., 1995). Both the P2X1 and P2X2 receptor subtypes are sensitive to inhibition by pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) and suramin. The pharmacological and kinetic properties of the P2X3 receptor from rat dorsal root ganglia (Chen et al., 1995; Lewis et al., 1995) and human heart (Garcia-Guzman et al., 1997b) are similar to those reported for the P2X1 receptor, including αβ-meATP sensitivity and rapid desensitization kinetics. The P2X4 receptor is non-desensitizing, αβ-meATP sensitive and has been identified in rat hippocampus (Bo et al., 1995), rat superior cervical ganglion (Buell et al., 1996) and human brain (Garcia-Guzman et al., 1997a). The rat and human homologs of the P2X4 receptor appear to differ in their sensitivity to suramin and PPADS, where the human P2X4 receptor is weakly sensitive and the rat P2X4 receptor is insensitive to these putative inhibitors (Garcia-Guzman et al., 1997a). The P2X7 receptor is distinguished by its ability to form a large pore upon prolonged or repeated agonist stimulation (Rassendren et al., 1997). P2X7 is partially activated by saturating concentrations of ATP, whereas it is fully activated by the synthetic ATP analog benzoylbenzoic ATP (BzATP, 2′ and 3′ mixed isomers) (Gargett et al., 1997).
The P2X2 and P2X3 receptor subtypes have been shown to form functional heteromeric receptors in native tissues (Lewis et al., 1995). The P2X2/3 heteromeric receptor appears to combine the pharmacological properties of P2X3 (αβ-meATP sensitivity) with the kinetic properties of P2X2 (slow desensitization) (Lewis et al., 1995; Radford et al., 1997), thereby facilitating its detection in situ or in heterologous expression systems.
To date, two additional P2X receptor subtypes have been identified, P2X5 and P2X6 (Collo et al., 1996; Garcia-Guzman et al., 1996). However, the P2X5 receptor mediates a very weak response to nucleotide agonists such that receptor-mediated changes in Ca2+ influx cannot be reliably measured. In contrast, the P2X6 receptor mediates a potent response to ATP, but is poorly expressed in heterologous cell systems (Collo et al., 1996). In vivo, these receptors may not exist as homomeric receptors, but rather as heteromers with other P2X receptor subtypes. Recently, the P2X4 and P2X6 receptor subtypes have been shown to form functional heteromers in vitro (Le et al., 1998).
Attempts to characterize endogenous and recombinant P2X receptor subtypes using radioligand binding techniques have met with limited success, primarily due to a lack of selective radioligands. Recent reports have called into question the binding specificity of a number of putative P2 receptor-selective radioligands, including []deoxyadenosine 5′-O-(1-thiotriphosphate) (dATPαS) (Schachter and Harden, 1997), []adenosine 5′-O-(2-thiodiphosphate) (ADPβS), and []adenosine 5′-O-(3-thiotriphosphate) (ATPγS) (Bianchi et al., 1998). These reports reveal no correlation between radioligand binding profiles and the functional activity of various P2 ligands, and thus conclude that radiolabeled nucleotide analogs are unsuitable for identifying and discriminating between P2 receptor subtypes expressed at physiological levels. The lack of P2 receptor subtype-selective ligands has complicated the characterization of these receptors in situ.
To provide a direct comparative pharmacological characterization of the P2X receptors, we have cloned the rat P2X2 and P2X3 receptors, and the human P2X1, P2X3, P2X4 and P2X7 receptors. Each P2X receptor subtype was functionally expressed in its homomeric form in stably transfected 1321N1 human astrocytoma cells, which have previously been shown to be devoid of endogenous P2X or P2Y receptor function (Bianchi et al., 1998). The rat P2X2 and P2X3 receptor subunits were co-expressed to form the functional P2X2/3 heteromeric receptor. The functional and pharmacological properties of P2X receptor-mediated Ca2+ influx were characterized in each cell line using a fluorescence based Ca2+ influx assay. The resulting pharmacological profiles were unique to each cell line and serve as a useful tool in the identification and characterization of endogenously expressed P2X receptors, while further emphasizing the need for P2 receptor subtype-selective ligands.
Section snippets
Materials
ATP, 2-methylthio-ATP (2-meS-ATP), αβ-meATP, suramin, and PPADS were obtained from Research Biochemicals International (Natick, MA). BzATP, ATPγS, ADP, UTP, and diadenosine polyphosphates (APnA, where n=3–6) were obtained from Sigma (St. Louis, MO). ADPβS and G418 were obtained from Calbiochem-Novabiochem (La Jolla, CA). Dulbecco's modified Eagle's medium (D-MEM) (with 4.5 mg ml−1 glucose and 4 mM l-glutamine) and fetal bovine serum were obtained from Hyclone Laboratories (Logan, UT).
Kinetics of ATP-activated P2X receptor responses
Activation of each P2X receptor subtype leads to a rapid increase in cytosolic Ca2+ levels (Fig. 1). The kinetics of Ca2+ influx is reflective of the electrophysiological response for each P2X receptor subtype (Fig. 2). The kinetics of the ligand-activated transmembrane currents, in turn, are consistent with previously reported data (see references in Section 1). Briefly, hP2X1 and hP2X3 receptors exhibit rapid desensitization kinetics whereas rP2X2, hP2X4, rP2X2/3 and hP2X7 desensitize slowly,
Discussion
P2X receptor subtypes were expressed and functionally characterized in a cellular background devoid of endogenous P2 receptor activity (1321N1 human astrocytoma cells). The kinetics of receptor activation, as measured by electrophysiological methods and by Ca2+ influx, are consistent with previously reported observations (see references in Section 1). hP2X1 and hP2X3 receptors exhibit rapid desensitization kinetics, whereas rP2X2, rP2X2/3, hP2X4 and hP2X7 receptors desensitize slowly, if at
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