J. Rettinger et al., Roles of individual N-glycans for ATP potency and expression of the rat P2X(1) receptor, J BIOL CHEM, 275(43), 2000, pp. 33542-33547
P2X(1) receptor subunits assemble in the ER of Xenopus oocytes to homotrime
rs that appear as ATP-gated cation channels at the cell surface. Here we ad
dress 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 in
stead 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 m
utants migrate as homotrimers when resolved by blue native PAGE. Recording
of ATP-gated currents reveals that elimination of Asn(153) or Asn(210) dimi
nishes 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, respective
ly. We conclude that at least one N-glycan per subunit of either position i
s absolutely required for the formation of P2X(1) receptors and that indivi
dual N-glycans possess marked positional effects on expression levels (Asn(
154), Asn(210)) and ATP potency (Asn(210)).