We have produced the putative extracellular domain (ECD) of the ATP-gated ion channel, P2X(2), in a bacterial expression system. The hexahistidine-tagged protein was purified by immobilized metal affinity chromatography and refolded by sulfitolysis and dialysis. We demonstrate that P2X(2)-ECD forms a stable tetramer in solution by gel filtration chromatography, dynamic light scattering and analytical sedimentation centrifugation. [alpha-P-32]ATP has been covalently cross-linked by UV irradiation to the P2X(2)-ECD and this binding is specific and competable by antagonists suramin and cibacron blue. These results indicate that the binding affinity among P2X(2)-ECD subunits is appreciably stronger than 3.4 mu M (0.1 mg/ml), implying that the extracellular domain of P2X(2) is primarly responsible for tetramerization of whole P2X(2) and thus probably plays a role in determining home-and heteromerization specificity of P2X channel subunits. (C) 1997 Academic Press.