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Molecular Pharmacology

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Research ArticleArticle

Radiolabeling of the Rat P2X4 Purinoceptor: Evidence for Allosteric Interactions of Purinoceptor Antagonists and Monovalent Cations with P2X Purinoceptors

Anton D. Michel, Kerry J. Miller, Kenneth Lundström, Gary N. Buell and Patrick P.A. Humphrey
Molecular Pharmacology March 1997, 51 (3) 524-532;
Anton D. Michel
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Kerry J. Miller
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Kenneth Lundström
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Gary N. Buell
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Patrick P.A. Humphrey
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Abstract

The rat recombinant P2X4 purinoceptor was expressed in CHO-K1 cells, and binding studies were performed using the radioligand [35S]adenosine-5′-O-(3-thio)triphosphate ([35S]ATPγS). In 50 mm Tris/1 mm EDTA assay buffer, pH 7.4 at 4°, [35S]ATPγS bound with high affinity to the P2X4 purinoceptor (KD= 0.13 nm, Bmax = 151 pmol/mg of protein). The purinoceptor agonists ATP and 2-methylthioadenosine triphosphate possessed nanomolar affinity for the P2X4 purinoceptor, whereas the antagonist suramin possessed much lower affinity (IC50 = 0.5 mm). Cibacron blue was more potent than suramin but produced a biphasic competition curve, whereas d-tubocurarine potentiated binding at concentrations in excess of 10 μm. The complex effects of cibacron blue and d-tubocurarine seemed to be due to an allosteric interaction with the P2X4purinoceptor because these compounds affected radioligand dissociation, measured after isotopic dilution with unlabeled ATPγS. Cibacron blue (1–100 μm) and d-tubocurarine (0.1–1 mm) produced rapid (10 sec to 5 min) decreases or increases, respectively, in the level of [35S]ATPγS binding measured immediately after initiation of the dissociation reaction. However, the subsequent rates of radioligand dissociation were not markedly different from those measured in their absence. Monovalent cations produced similar affects on the P2X4purinoceptor and, like d-tubocurarine, increased [35S]ATPγS binding. The actions ofd-tubocurarine and sodium were not additive. The findings from this study indicate that [35S]ATPγS can be used to label the P2X4 purinoceptor and suggest that this binding can be enhanced by monovalent cations andd-tubocurarine and may be subject to negative allosteric modulation to varying degrees by different purinoceptor antagonists.

Footnotes

  • Send reprint requests to: Dr. A. D. Michel, Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge, Cambridge CB2 1QJ, UK. E-mail:adm7393{at}gcr.co.uk

  • ↵1 A. D. Michel, unpublished observations.

  • ↵2 A. D. Michel and K. J. Miller, unpublished observations.

  • ↵3 A. D. Michel and K. J. Miller, unpublished observations.

  • Abbreviations:
    [35S]ATPγS
    [35S]adenosine-5′-O-(3-thio)triphosphate
    2-Me-S-ATP
    2-methylthioadenosine triphosphate
    αβ-MeADP
    α,β-methylene ADP
    αβ-MeATP
    α,β-methylene ATP
    βγ-MeATP β
    γ-methylene ATP
    DIDS
    4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid
    P5P
    pyridoxal-5′-phosphate
    PPADS
    pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid
    SFV
    Semliki forest virus
    CHO
    Chinese hamster ovary
    HEPES
    4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
    • Received August 12, 1996.
    • Accepted November 11, 1996.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology
Vol. 51, Issue 3
1 Mar 1997
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Research ArticleArticle

Radiolabeling of the Rat P2X4 Purinoceptor: Evidence for Allosteric Interactions of Purinoceptor Antagonists and Monovalent Cations with P2X Purinoceptors

Anton D. Michel, Kerry J. Miller, Kenneth Lundström, Gary N. Buell and Patrick P.A. Humphrey
Molecular Pharmacology March 1, 1997, 51 (3) 524-532;

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Research ArticleArticle

Radiolabeling of the Rat P2X4 Purinoceptor: Evidence for Allosteric Interactions of Purinoceptor Antagonists and Monovalent Cations with P2X Purinoceptors

Anton D. Michel, Kerry J. Miller, Kenneth Lundström, Gary N. Buell and Patrick P.A. Humphrey
Molecular Pharmacology March 1, 1997, 51 (3) 524-532;
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