P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels.
P2X receptors are cation channels gated by extracellular ATP. The seven known P2X isoforms possess no sequence homology with other proteins. Here we studied the quaternary structure of P2X receptors by chemical cross-linking and blue native PAGE. P2X1 and P2X3 were N-terminally tagged with six histidine residues to allow for non-denaturing receptor isolation from cRNA-injected, [35S]methionine-labeled oocytes. The His-tag did not change the electrophysiological properties of the P2X1 receptor. His-P2X1 was found to carry four N-glycans per polypeptide chain, only one of which acquired Endo H resistance en route to the plasma membrane. 3, 3'-Dithiobis(sulfosuccinimidylpropionate) (DTSSP) and two of three bifunctional analogues of the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) cross-linked digitonin-solubilized His-P2X1 and His-P2X3 quantitatively to homo-trimers. Likewise, when analyzed by blue native PAGE, P2X receptors purified in digitonin or dodecyl-beta-D-maltoside migrated entirely as non-covalently linked homo-trimers, whereas the alpha2 beta gamma delta nicotinic acetylcholine receptor (used as a positive control) migrated as the expected pentamer. P2X monomers remained undetected soon after synthesis, indicating that trimerization occurred in the endoplasmic reticulum. The plasma membrane form of His-P2X1 was also identified as a homo-trimer. If n-octylglucoside was used for P2X receptor solubilization, homo-hexamers were observed, suggesting that trimers can aggregate to form larger complexes. We conclude that trimers represent an essential element of P2X receptor structure.


BACKGROUND
blue native PAGE/cross-linking/P2X receptor/quaternary structure.
Nicke
A
A
Biocenter of the Johann Wolfgang Goethe-University of Frankfurt, Frankfurt, Germany.
Bäumert
H G
HG
Rettinger
J
J
Eichele
A
A
Lambrecht
G
G
Mutschler
E
E
Schmalzing
G
G
D000818:Animals; D003432:Cross-Linking Reagents; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides / Q000737:chemistry; D006031:Glycosylation / Q000187:drug effects; D006596:Hexosaminidases / Q000494:pharmacology; D006639:Histidine / Q000235:genetics; D007473:Ion Channels / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology; D009865:Oocytes / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D011950:Receptors, Cholinergic / Q000096:biosynthesis / Q000737:chemistry; D018048:Receptors, Purinergic P2 / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D058469:Receptors, Purinergic P2X; D058477:Receptors, Purinergic P2X3; D014982:Xenopus laevis
D003432:Cross-Linking Reagents; D005960:Glucosides; D007473:Ion Channels; D008024:Ligands; D008565:Membrane Proteins; D058919:Purinergic P2 Receptor Antagonists; D011950:Receptors, Cholinergic; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D058477:Receptors, Purinergic P2X3; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; C018619:octyl-beta-D-glucoside; D006639:Histidine; D011732:Pyridoxal Phosphate; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase