N-Glycans mutations rule oligomeric assembly and functional expression of P2X 3 receptor for extracellular ATP

N-Glycosylation affects the function of ion channels at the level of multisubunit assembly, protein trafficking, ligand binding and channel opening. Like the majority of membrane proteins, ionotropic P2X receptors for extracellular ATP are glycosylated in their extracellular moiety. Here, we used site-directed mutagenesis to the four predicted N-glycosylation sites of P2X3 receptor (Asn ¹³⁹, Asn ¹⁷⁰, Asn ¹⁹⁴ and Asn ²⁹⁰) and performed comparative analysis of the role of N-glycans on protein stability, plasma membrane delivery, trimer formation and inward currents. We have found that in transiently transfected HEK293 cells, Asn ¹⁷⁰ is apparently the most important site for receptor stability, since its mutation causes a primary loss in protein content and indirect failure in membrane expression, oligomeric association and inward current responses. Even stronger effects are obtained when mutating Thr ¹⁷² in the same glycosylation consensus. Asn ¹⁹⁴ and Asn ²⁹⁰ are the most dispensable, since even their simultaneous mutation does not affect any tested receptor feature. All double mutants containing Asn ¹⁷⁰ mutation or the Asn ¹³⁹/Asn ²⁹⁰ double mutant are instead almost unable to assemble into a functional trimeric structure. The main emerging finding is that the inability to assemble into trimers might account for the impaired function in P2X3 mutants where residue Asn ¹⁷⁰ is replaced. These results improve our knowledge about the role of N-glycosylation in proper folding and oligomeric association of P2X3 receptor.