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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-β-D-maltoside migrated entirely as non-covalently linked homo-trimers, whereas the α2βγδ 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. [ABSTRACT FROM AUTHOR]

1. ATP γP-imidazolidate, synthesized from ATP and carbonyldiimidazole, inhibits the Ca2+-ATPase of sarcoplasmic reticulum in a biphasic manner. A fast first phase is concentration-dependent while a slower second phase is independent of the inhibitor concentration. 2. The inhibition is calcium- and magnesium-dependent. No inhibition occurs in the absence of either cation. 3. Inhibition of the Ca2+-ATPase can be prevented by the protection with ATP. 4. The loss of ATPase activity is pH-dependent. Maximal inhibition coincides with maximal ATPase activity. It indicates a participation of the reacting amino acid side chain in the catalytic cycle. 5. The incorporation of radioactive inhibitor is reversed in a time-dependent fashion while the Ca2+-ATPase remains inhibited. 6. We conclude that ATP γP-imidazolidate initially reacts covalently with an amino acid side chain, probably Asp-351, but is subsequently expelled by a reaction with a second amino acid. 7. This two-step reaction induces an intramolecular cross-link which can be shown by the creation of a new protein band on SDS-PAGE which originates in the Ca2+-ATPase. [ABSTRACT FROM AUTHOR]