Roles of individual N-glycans for ATP potency and expression of the rat P2X(1) receptor

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)).