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P2X 1 and P2X 3 receptors form stable trimers: a novel structural motif of ligand‐gated ion channels
Author(s) -
Nicke Annette,
Bäumert Hans G.,
Rettinger Jürgen,
Eichele Annette,
Lambrecht Günter,
Mutschler Ernst,
Schmalzing Günther
Publication year - 1998
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/17.11.3016
Subject(s) - biology , receptor , motif (music) , ion channel , structural motif , biophysics , ligand gated ion channel , biochemistry , microbiology and biotechnology , physics , acoustics
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. P2X 1 and P2X 3 were N‐terminally tagged with six histidine residues to allow for non‐denaturing receptor isolation from cRNA‐injected, [ 35 S]methionine‐labeled oocytes. The His‐tag did not change the electrophysiological properties of the P2X 1 receptor. His‐P2X 1 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′‐Dithio bis (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‐P2X 1 and His‐P2X 3 quantitatively to homo‐trimers. Likewise, when analyzed by blue native PAGE, P2X receptors purified in digitonin or dodecyl‐β‐ D ‐maltoside migrated entirely as non‐covalently linked homo‐trimers, whereas the α 2 βγδ 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‐P2X 1 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.