Roles of individual N-glycans for ATP potency and expression of the rat P2X1 receptor.
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)).
Rettinger
J
J
Department of Pharmacology, Johann Wolfgang Goethe University, Biocenter N 260, Marie-Curie-Strasse 9, D-60439 Frankfurt/Main, Germany.
Aschrafi
A
A
Schmalzing
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D006031:Glycosylation; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D021122:Protein Subunits; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
D011134:Polysaccharides; D021122:Protein Subunits; D018048:Receptors, Purinergic P2; D000255:Adenosine Triphosphate