Roles of individual N-glycans for ATP potency and expression of the rat P2X1 receptor

J Biol Chem. 2000 Oct 27;275(43):33542-7. doi: 10.1074/jbc.M002918200.


P2X(1) receptor subunits assemble in the ER of Xenopus oocytes to homotrimers that appear as ATP-gated cation channels at the cell surface. Here we address the extent to which N-glycosylation contributes to assembly, surface appearance, and ligand recognition of P2X(1) receptors. SDS-polyacrylamide gel electrophoresis (PAGE) analysis of glycan minus mutants carrying Gln instead of Asn at five individual NXT/S sequons reveals that Asn(284) remains unused because of a proline in the +4 position. The four other sites (Asn(153), Asn(184), Asn(210), and Asn(300)) carry N-glycans, but solely Asn(300) located only eight residues upstream of the predicted reentry loop of P2X(1) acquires complex-type carbohydrates. Like parent P2X(1), glycan minus mutants migrate as homotrimers when resolved by blue native PAGE. Recording of ATP-gated currents reveals that elimination of Asn(153) or Asn(210) diminishes or increases functional expression levels, respectively. In addition, elimination of Asn(210) causes a 3-fold reduction of the potency for ATP. If three or all four N-glycosylation sites are simultaneously eliminated, formation of P2X(1) receptors is severely impaired or abolished, respectively. We conclude that at least one N-glycan per subunit of either position is absolutely required for the formation of P2X(1) receptors and that individual N-glycans possess marked positional effects on expression levels (Asn(154), Asn(210)) and ATP potency (Asn(210)).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Amino Acid Sequence
  • Animals
  • Glycosylation
  • Molecular Sequence Data
  • Polysaccharides / chemistry
  • Polysaccharides / metabolism*
  • Protein Subunits
  • Rats
  • Receptors, Purinergic P2 / chemistry*
  • Receptors, Purinergic P2 / metabolism
  • Structure-Activity Relationship
  • Xenopus laevis


  • Polysaccharides
  • Protein Subunits
  • Receptors, Purinergic P2
  • Adenosine Triphosphate