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

Biochem Biophys Res Commun. 1997 Nov 26;240(3):618-22. doi: 10.1006/bbrc.1997.7713.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Animals
  • Cell Membrane / chemistry
  • Chromatography, Gel
  • Cross-Linking Reagents
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Gene Expression
  • Ion Channels / chemistry
  • Ion Channels / isolation & purification
  • Molecular Weight
  • Protein Conformation*
  • Protein Folding
  • Rats
  • Receptors, Purinergic P2 / chemistry*
  • Receptors, Purinergic P2 / genetics
  • Receptors, Purinergic P2 / isolation & purification
  • Receptors, Purinergic P2 / metabolism
  • Receptors, Purinergic P2X2
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Suramin / pharmacology
  • Triazines / pharmacology
  • Ultracentrifugation
  • Ultraviolet Rays


  • Cross-Linking Reagents
  • Ion Channels
  • Receptors, Purinergic P2
  • Receptors, Purinergic P2X2
  • Recombinant Proteins
  • Triazines
  • Cibacron Blue F 3GA
  • Suramin
  • Adenosine Triphosphate