Characterisation of a recombinant P2Y purinoceptor

We have previously cloned a cDNA encoding a G-protein-coupled P₂ purinoceptor from chick brain and designated this as a P_2Y1 purinoceptor (Webb, T.E., J. Simon, B.J. Krishek, A.N. Bateson, T.G. Smart, B.J. King, G. Burnstock and E.A. Barnard, 1993, FEBS Lett. 324, 219). Here, we describe the further characterisation of this recombinant receptor expressed in both simian kidney endothelial (COS-7) cells and Xenopus oocytes. In transfected COS-7 cell membranes, the recombinant receptor showed a high level of expression ($\text{B}{}_{\mathrm{<mtext>max</mtext>}}\text{= 7.9 ± 2.2}\text{pmol}\text{[}{}^{35}\text{S}\text{]}\text{dATP}\text{α}\text{S bound/mg protein}$) and affinity (K_d = 6.6 ± 0.3 nM). In these COS-7 cells, the activation of the implanted purinoceptor induced a suramin-sensitive formation of inositol 1,4,5-triphosphate (1,4,5InsP₃). Upon expression in Xenopus oocytes, ATP was the only natural nucleoside triphosphate to elicit a Ca²⁺-activated chloride current. The P₂ purinoceptor antagonists suramin and Reactive Blue-2 were both able to inhibit this evoked current. Utilizing both expression systems, the binding affinity profile and the functional pharmacological profile of the agonists, the common series found was: 2-methylthioATP (2-MeSATP) ⩾ ATP > ADPβS > ADP. These two agonist series and the lack of activity of adenosine, α,β-methyleneATP (α,β-meATP), 3′-O-(4- benzoyl)benzoyl-ATP (Bz-ATP) and UTP, together confirmed that this receptor is a specific subtype of the P_2Y purinoceptors.