P2X(1) AND P2X(3) RECEPTORS FORM STABLE TRIMERS - A NOVEL STRUCTURAL MOTIF OF LIGAND-GATED ION CHANNELS

P2X receptors are cation channels gated by extracellular ATP. The seve n 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. P2X(1) and P2X(3) were N-terminal ly tagged with six histidine residues to allow for non-denaturing rece ptor isolation from cRNA-injected, [S-35]methionine-labeled oocytes. T he His-tag did not change the electrophysiological properties of the P 2X(1) receptor. His-P2X(1) was found to carry four N-glycans per polyp eptide chain, only one of which acquired Endo H resistance en route to the plasma membrane. 3,3'-Dithiobis(sulfosuccinimidylpropionate) (DTS SP) and two of three bifunctional analogues of the P2X receptor antago nist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) cro ss-linked digitonin-solubilized His-P2X(1) and His-P2X(3) quantitative ly to homo-trimers. Likewise, when analyzed by blue native PAGE, P2X r eceptors purified in digitonin or dodecyl-beta-D-maltoside migrated en tirely as non-covalently linked homo-trimers, whereas the alpha(2) bet a gamma delta nicotinic acetylcholine receptor (used as a positive con trol) migrated as the expected pentamer, P2X monomers remained undetec ted soon after synthesis, indicating that trimerization occurred in th e endoplasmic reticulum. The plasma membrane form of His-P2X(1) was al so identified as a homo-trimer, If n-octylglucoside was used for P2X r eceptor solubilization, homo-hexamers were observed, suggesting that t rimers can aggregate to form larger complexes. We conclude that trimer s represent an essential element of P2X receptor structure.