title: Radiolabeling of the rat P2X4 purinoceptor: evidence for allosteric interactions of purinoceptor antagonists and monovalent cations with P2X purinoceptors.
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]ATPgammaS). In 50 mM Tris/1 mM EDTA assay buffer, pH 7.4 at 4 degrees, [35S]ATPgammaS 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 microM. The complex effects of cibacron blue and d-tubocurarine seemed to be due to an allosteric interaction with the P2X4 purinoceptor because these compounds affected radioligand dissociation, measured after isotopic dilution with unlabeled ATPgammaS. Cibacron blue (1-100 microM) and d-tubocurarine (0.1-1 mM) produced rapid (10 sec to 5 min) decreases or increases, respectively, in the level of [35S]ATPgammaS 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 P2X4 purinoceptor and, like d-tubocurarine, increased [35S]ATPgammaS binding. The actions of d-tubocurarine and sodium were not additive. The findings from this study indicate that [35S]ATPgammaS can be used to label the P2X4 purinoceptor and suggest that this binding can be enhanced by monovalent cations and d-tubocurarine and may be subject to negative allosteric modulation to varying degrees by different purinoceptor antagonists.
authors.affiliation: Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge, UK. adm7393@gcr.co.uk
authors.forename: A D
authors.initials: AD
authors.lastname: Michel
authors.forename: K J
authors.initials: KJ
authors.lastname: Miller
authors.forename: K
authors.initials: K
authors.lastname: Lundström
authors.forename: G N
authors.initials: GN
authors.lastname: Buell
authors.forename: P P
authors.initials: PP
authors.lastname: Humphrey
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D007700:Kinetics; D011485:Protein Binding; D058914:Purinergic Antagonists; D051381:Rats; D011983:Receptors, Purinergic / Q000737:chemistry*; D011994:Recombinant Proteins / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013462:Sulfur Radioisotopes; D014162:Transfection; D014403:Tubocurarine / Q000494:pharmacology
chemical_list: D000345:Affinity Labels; D058914:Purinergic Antagonists; D011983:Receptors, Purinergic; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D014403:Tubocurarine