Radiolabeling of the rat P2X(4) purinoceptor: Evidence for allosteric interactions of purinoceptor antagonists and monovalent cations with P2X purinoceptors

The rat recombinant P2X(4) purinoceptor was expressed in CHO-K1 cells, and binding studies were performed using the radioligand [S-35]adenosine-5'-O-(3-thio)triphosphate ([S-35]ATP gamma S). In 50 mM Tris/1 mM EDTA assay buffer, pH 7.4 at 4 degrees, [S-35]ATP gamma S bound with high affinity to the P2X(4) purinoceptor (K-D = 0.13 nM, B-max = 151 pmol/mg of protein). The purinoceptor agonists ATP and 2-methylthioadenosine triphosphate possessed nanomolar affinity for the P2X(4) 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 mu M. The complex effects of cibacron blue and d-tubocurarine seemed to be due to an allosteric interaction with the P2X(4) purinoceptor because these compounds affected radioligand dissociation, measured after isotopic dilution with unlabeled ATP gamma S. Cibacron blue (1-100 mu M) and d-tubocurarine (0.1-1 mM) produced rapid (10 sec to 5 min) decreases or increases, respectively, in the level of [S-35]ATP gamma S 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 P2X(4) purinoceptor and, like d-tubocurarine, increased [S-35]ATP gamma S binding. The actions of d-tubocurarine and sodium were not additive. The findings from this study indicate that [S-35]ATP gamma S can be used to label the P2X(4) 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.