Abstract
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
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Triphosphate / metabolism*
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Animals
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Cell Membrane / chemistry
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Chromatography, Gel
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Cross-Linking Reagents
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Electrophoresis, Polyacrylamide Gel
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Escherichia coli / genetics
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Gene Expression
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Ion Channels / chemistry
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Ion Channels / isolation & purification
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Molecular Weight
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Protein Conformation*
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Protein Folding
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Rats
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Receptors, Purinergic P2 / chemistry*
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Receptors, Purinergic P2 / genetics
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Receptors, Purinergic P2 / isolation & purification
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Receptors, Purinergic P2 / metabolism
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Receptors, Purinergic P2X2
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Suramin / pharmacology
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Triazines / pharmacology
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Ultracentrifugation
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Ultraviolet Rays
Substances
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Cross-Linking Reagents
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Ion Channels
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Receptors, Purinergic P2
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Receptors, Purinergic P2X2
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Recombinant Proteins
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Triazines
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Cibacron Blue F 3GA
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Suramin
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Adenosine Triphosphate