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 (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 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.