P2X receptors are cation channels gated by extracellular ATP. The seven known P2X isoforms possess no sequence homology with other proteins. Here we studied the quaternary structure of P2X receptors by chemical cross-linking and blue native PAGE. P2X 1 and P2X 3 were N-terminally tagged with six histidine residues to allow for non-denaturing receptor isolation from cRNA-injected, [ 35 S]methionine-labeled oocytes. The His-tag did not change the electrophysiological properties of the P2X 1 receptor. His-P2X 1 was found to carry four N-glycans per polypeptide chain, only one of which acquired Endo H resistance en route to the plasma membrane. 3,3Ј-Dithiobis(sulfosuccinimidylpropionate) (DTSSP) and two of three bifunctional analogues of the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2Ј,4Ј-disulfonic acid (PPADS) cross-linked digitonin-solubilized His-P2X 1 and His-P2X 3 quantitatively to homo-trimers. Likewise, when analyzed by blue native PAGE, P2X receptors purified in digitonin or dodecyl-β-D-maltoside migrated entirely as non-covalently linked homo-trimers, whereas the α 2 βγδ nicotinic acetylcholine receptor (used as a positive control) migrated as the expected pentamer. P2X monomers remained undetected soon after synthesis, indicating that trimerization occurred in the endoplasmic reticulum. The plasma membrane form of His-P2X 1 was also identified as a homo-trimer. If n-octylglucoside was used for P2X receptor solubilization, homo-hexamers were observed, suggesting that trimers can aggregate to form larger complexes. We conclude that trimers represent an essential element of P2X receptor structure.

No abstract

P2X receptors are nucleotide-gated cation channels composed of homomeric or heteromeric assemblies of three subunits. In the past 7 years, an extended series (P2X1-7) of P2X subunits has been cloned from vertebrate tissues. In this rapidly expanding field, one of the main current challenges is to relate the cloned P2X receptor subtypes to the diverse physiological responses mediated by the native P2X receptors. However, the paucity of useful ligands, especially subtype-selective agonists and antagonists as well as radioligands, acts as a considerable impediment to progress. Most of the ligands available are highly limited in terms of their kinetics of action, receptor-affinity, subtype-selectivity and P2X receptor-specificity. Their suspected ability to be a substrate for ecto-nucleotidases or to inhibit these enzymes also complicates their use. A number of new antagonists at P2X receptors have recently been described which to some degree are more potent and more selective than earlier antagonists like suramin or pyridoxal-5'-phosphate-6-azophenyl 2',4'-disulfonate (PPADS). This work moves us closer to the ideal goal of classifying the recombinant and native P2X receptor subtypes on the basis of antagonist profiles. This review begins with a brief account of the current status of P2X receptors. It then focuses on the pharmacological properties of a series of key P2 receptor agonists and antagonists and will finish with the discussion of some related therapeutic possibilities.

1 Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), an inhibitor of P2x-purinoceptormediated responses in rabbit vas deferens, was investigated for its ability to antagonize contractions evoked by a,B-methylene ATP (a,P-MeATP), carbachol and electrical field stimulation in the rabbit urinary bladder detrusor muscle.2 PPADS (1-30 gM) caused concentration-dependent inhibition of contractions to the stable P2X-purinoceptor agonist, cx,,-MeATP, decreasing the maximum response to a,4-MeATP (30 JAM) at concentrations of 3-30 JiM. The pD2 value for a,4-MeATP in the absence of PPADS was 6.52 ± 0.10 (8). In the presence of PPADS at concentrations of 1, 3, 10 and 30 gM the negative log concentrations of a,4-MeATP that cause the same contractile response as the pD2 value were significantly different from control, being respectively 6.17 ± 0.09 (8), 5.64 ± 0.12 (7), 5.15 ± 0.23 (7) and 4.78 ± 0.22 (5). 3 PPADS (1-30IM) caused concentration-dependent inhibition of contractions to stimulation of intramural purinergic nerves (1 -32 Hz). There was a greater inhibition at lower frequencies (1-8 Hz)than at higher frequencies (16-32 Hz). PPADS, 30 JAM, did not produce significantly greater antagonism than 1OIJM. 4 PPADS (30 jAM) had no significant influence on the contractile potency of carbachol: the pD2 values of carbachol in the absence and presence of PPADS were not significantly different being 6.42 ± 0.16 (5) and 6.33 ± 0.18 (5), respectively. However, PPADS caused a small, but significant, suppression of the maximal response of carbachol, reducing it by approximately 9%. 6 Thus, PPADS (1-30 JM) antagonized responses mediated via P2X-purinoceptors in the rabbit urinary bladder. It was selective for P2-purinoceptor-mediated contractions rather than those mediated via muscarinic receptors. Binding studies demonstrated that the antagonistic effect of PPADS is via a direct interaction with P2x-purinoceptors.

No abstract