Molecular assembly of the extracellular domain of P2X2, an ATP-gated ion channel.

We have produced the putative extracellular domain (ECD) of the ATP-gated ion channel, P2X2, 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 P2X2-ECD forms a stable tetramer in solution by gel filtration chromatography, dynamic light scattering and analytical sedimentation centrifugation. [alpha-32P]ATP has been covalently cross-linked by UV irradiation to the P2X2-ECD and this binding is specific and competable by antagonists suramin and cibacron blue. These results indicate that the binding affinity among P2X2-ECD subunits is appreciably stronger than 3.4 microM (0.1 mg/ml), implying that the extracellular domain of P2X2 is primarly responsible for tetramerization of whole P2X2 and thus probably plays a role in determining homo- and heteromerization specificity of P2X channel subunits.