Skip to main content
PMC home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 1996 Aug;118(7):1806–1812. doi: 10.1111/j.1476-5381.1996.tb15607.x

A comparison of the binding characteristics of recombinant P2X1 and P2X2 purinoceptors.

A D Michel 1, K Lundström 1, G N Buell 1, A Surprenant 1, S Valera 1, P P Humphrey 1
PMCID: PMC1909833  PMID: 8842447

Abstract

1. We have recently provided evidence that [35S]-adenosine 5'-O-[3-thiotriphosphate] ([35S]-ATP gamma S) can label the human bladder recombinant P2X1 purinoceptor (human P2X1 purinoceptor). In this study we have characterized the binding of [35S]-ATP gamma S to a second P2X purinoceptor subtype, the rat PC12 phaeochromocytoma cell recombinant P2X2 purinoceptor (rat P2X2 purinoceptor), and compared its binding properties with those of both endogenous and recombinant P2X1 purinoceptors. 2. Infection of CHO-K1 cells with the rat P2X2 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]-ATP gamma S but not for [3H]-alpha, beta-methylene ATP ([3H]-alpha beta meATP). 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 P2X2 purinoceptor can be labelled using [35S]-ATP gamma S. 3. The binding characteristics of the rat P2X2 purinoceptor were compared with those of the human P2X1 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 alpha, beta-methylene ATP (alpha beta meATP). Thus, in the absence of divalent cations, alpha beta meATP possessed low affinity for both the human P2X1 purinoceptor (pIC50 = 7.2) and rat P2X2 purinoceptor (pIC50 = 7.1) labelled using [35S]-ATP gamma S. However, when the recombinant P2X purinoceptors were labelled with [3H]-alpha beta meATP in the presence of 4 mM CaCl2, the affinity of alpha beta meATP for the human P2X1 purinoceptor increased (pIC50 for alpha beta meATP = 8.2), while the affinity of the rat P2X2 purinoceptor for alpha beta meATP did not change (pIC50 for alpha beta meATP = 6.8). 4. Affinity estimates of 15 other nucleotide analogues for the [35S]-ATP gamma S 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 P2X2 than for human P2X1 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 P2X1 than for the rat P2X2 purinoceptor. These data provide a correlation coefficient (r) of 0.894. 5. There was some evidence for species differences in the P2X1 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 P2X1 purinoceptors than for the human recombinant P2X1 purinoceptor. These differences were reflected in a slightly lower correlation coefficient, when comparing across species between the human recombinant P2X1 purinoceptor and the endogenous P2X1 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 P2X1 purinoceptors (r = 0.995). 6. In summary, [35S]-ATP gamma S can be used to label the recombinant P2X1 and P2X2 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.

Full text

PDF
1806

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abbracchio M. P., Burnstock G. Purinoceptors: are there families of P2X and P2Y purinoceptors? Pharmacol Ther. 1994;64(3):445–475. doi: 10.1016/0163-7258(94)00048-4. [DOI] [PubMed] [Google Scholar]
  2. Bo X., Zhang Y., Nassar M., Burnstock G., Schoepfer R. A P2X purinoceptor cDNA conferring a novel pharmacological profile. FEBS Lett. 1995 Nov 13;375(1-2):129–133. doi: 10.1016/0014-5793(95)01203-q. [DOI] [PubMed] [Google Scholar]
  3. Brake A. J., Wagenbach M. J., Julius D. New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor. Nature. 1994 Oct 6;371(6497):519–523. doi: 10.1038/371519a0. [DOI] [PubMed] [Google Scholar]
  4. Burnstock G., Kennedy C. Is there a basis for distinguishing two types of P2-purinoceptor? Gen Pharmacol. 1985;16(5):433–440. doi: 10.1016/0306-3623(85)90001-1. [DOI] [PubMed] [Google Scholar]
  5. Bültmann R., Starke K. Blockade by 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) of P2X-purinoceptors in rat vas deferens. Br J Pharmacol. 1994 Jun;112(2):690–694. doi: 10.1111/j.1476-5381.1994.tb13131.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen C. C., Akopian A. N., Sivilotti L., Colquhoun D., Burnstock G., Wood J. N. A P2X purinoceptor expressed by a subset of sensory neurons. Nature. 1995 Oct 5;377(6548):428–431. doi: 10.1038/377428a0. [DOI] [PubMed] [Google Scholar]
  7. Cheng Y., Prusoff W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol. 1973 Dec 1;22(23):3099–3108. doi: 10.1016/0006-2952(73)90196-2. [DOI] [PubMed] [Google Scholar]
  8. Evans R. J., Kennedy C. Characterization of P2-purinoceptors in the smooth muscle of the rat tail artery: a comparison between contractile and electrophysiological responses. Br J Pharmacol. 1994 Nov;113(3):853–860. doi: 10.1111/j.1476-5381.1994.tb17071.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Evans R. J., Lewis C., Buell G., Valera S., North R. A., Surprenant A. Pharmacological characterization of heterologously expressed ATP-gated cation channels (P2x purinoceptors). Mol Pharmacol. 1995 Aug;48(2):178–183. [PubMed] [Google Scholar]
  10. Fredholm B. B., Abbracchio M. P., Burnstock G., Daly J. W., Harden T. K., Jacobson K. A., Leff P., Williams M. Nomenclature and classification of purinoceptors. Pharmacol Rev. 1994 Jun;46(2):143–156. [PMC free article] [PubMed] [Google Scholar]
  11. Humphrey P. P., Buell G., Kennedy I., Khakh B. S., Michel A. D., Surprenant A., Trezise D. J. New insights on P2X purinoceptors. Naunyn Schmiedebergs Arch Pharmacol. 1995 Dec;352(6):585–596. doi: 10.1007/BF00171316. [DOI] [PubMed] [Google Scholar]
  12. Inoue R., Brading A. F. The properties of the ATP-induced depolarization and current in single cells isolated from the guinea-pig urinary bladder. Br J Pharmacol. 1990 Jul;100(3):619–625. doi: 10.1111/j.1476-5381.1990.tb15856.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Khakh B. S., Humphrey P. P., Surprenant A. Electrophysiological properties of P2X-purinoceptors in rat superior cervical, nodose and guinea-pig coeliac neurones. J Physiol. 1995 Apr 15;484(Pt 2):385–395. doi: 10.1113/jphysiol.1995.sp020672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Khakh B. S., Michel A., Humphrey P. P. Estimates of antagonist affinities at P2X purinoceptors in rat vas deferens. Eur J Pharmacol. 1994 Oct 3;263(3):301–309. doi: 10.1016/0014-2999(94)90726-9. [DOI] [PubMed] [Google Scholar]
  15. Khakh B. S., Surprenant A., Humphrey P. P. A study on P2X purinoceptors mediating the electrophysiological and contractile effects of purine nucleotides in rat vas deferens. Br J Pharmacol. 1995 May;115(1):177–185. doi: 10.1111/j.1476-5381.1995.tb16336.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lewis C., Neidhart S., Holy C., North R. A., Buell G., Surprenant A. Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons. Nature. 1995 Oct 5;377(6548):432–435. doi: 10.1038/377432a0. [DOI] [PubMed] [Google Scholar]
  17. Liljeström P., Garoff H. A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology (N Y) 1991 Dec;9(12):1356–1361. doi: 10.1038/nbt1291-1356. [DOI] [PubMed] [Google Scholar]
  18. Lundström K., Mills A., Buell G., Allet E., Adami N., Liljeström P. High-level expression of the human neurokinin-1 receptor in mammalian cell lines using the Semliki Forest virus expression system. Eur J Biochem. 1994 Sep 15;224(3):917–921. doi: 10.1111/j.1432-1033.1994.00917.x. [DOI] [PubMed] [Google Scholar]
  19. Michel A. D., Humphrey P. P. Distribution and characterisation of [3H]alpha,beta-methylene ATP binding sites in the rat. Naunyn Schmiedebergs Arch Pharmacol. 1993 Dec;348(6):608–617. doi: 10.1007/BF00167237. [DOI] [PubMed] [Google Scholar]
  20. Michel A. D., Humphrey P. P. Effects of metal cations on [3H]alpha,beta-methylene ATP binding in rat vas deferens. Naunyn Schmiedebergs Arch Pharmacol. 1994 Aug;350(2):113–122. doi: 10.1007/BF00241084. [DOI] [PubMed] [Google Scholar]
  21. Michel A. D., Humphrey P. P. High affinity P2x-purinoceptor binding sites for [35S]-adenosine 5'-O-[3-thiotriphosphate] in rat vas deferens membranes. Br J Pharmacol. 1996 Jan;117(1):63–70. doi: 10.1111/j.1476-5381.1996.tb15155.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Michel A. D., Lundström K., Buell G. N., Surprenant A., Valera S., Humphrey P. P. The binding characteristics of a human bladder recombinant P2X purinoceptor, labelled with [3H]-alpha beta meATP, [35S]-ATP gamma S or [33P]-ATP. Br J Pharmacol. 1996 Mar;117(6):1254–1260. doi: 10.1111/j.1476-5381.1996.tb16723.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Trezise D. J., Michel A. D., Grahames C. B., Khakh B. S., Surprenant A., Humphrey P. P. The selective P2X purinoceptor agonist, beta,gamma-methylene-L-adenosine 5'-triphosphate, discriminates between smooth muscle and neuronal P2X purinoceptors. Naunyn Schmiedebergs Arch Pharmacol. 1995 Jun;351(6):603–609. doi: 10.1007/BF00170159. [DOI] [PubMed] [Google Scholar]
  24. Valera S., Hussy N., Evans R. J., Adami N., North R. A., Surprenant A., Buell G. A new class of ligand-gated ion channel defined by P2x receptor for extracellular ATP. Nature. 1994 Oct 6;371(6497):516–519. doi: 10.1038/371516a0. [DOI] [PubMed] [Google Scholar]
  25. Valera S., Talabot F., Evans R. J., Gos A., Antonarakis S. E., Morris M. A., Buell G. N. Characterization and chromosomal localization of a human P2X receptor from the urinary bladder. Receptors Channels. 1995;3(4):283–289. [PubMed] [Google Scholar]
  26. de Meis L., Suzano V. A. Uncoupling of muscle and blood platelets Ca2+ transport ATPases by heparin. Regulation by K+. J Biol Chem. 1994 May 20;269(20):14525–14529. [PubMed] [Google Scholar]

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES