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.
Kim
M
M
Structural Biology Laboratory, Salk Institute for Biological Sciences, La Jolla, California 92037, USA.
Yoo
O J
OJ
Choe
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007473:Ion Channels / Q000737:chemistry / Q000302:isolation & purification; D008970:Molecular Weight; D011487:Protein Conformation; D017510:Protein Folding; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D058476:Receptors, Purinergic P2X2; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013498:Suramin / Q000494:pharmacology; D014227:Triazines / Q000494:pharmacology; D014461:Ultracentrifugation; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D007473:Ion Channels; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D011994:Recombinant Proteins; D014227:Triazines; C012842:Cibacron Blue F 3GA; D013498:Suramin; D000255:Adenosine Triphosphate