title: N-Glycans mutations rule oligomeric assembly and functional expression of P2X3 receptor for extracellular ATP.
abstract: 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 P2X(3) 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 P2X(3) 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 P2X(3) receptor.
authors.affiliation: Laboratory Cellular Neurobiology, Santa Lucia Foundation, Rome, Italy. fabrizio.vacca@unige.ch
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Vacca
authors.forename: Nadia
authors.initials: N
authors.lastname: D'Ambrosi
authors.forename: Valeria
authors.initials: V
authors.lastname: Nestola
authors.forename: Susanna
authors.initials: S
authors.lastname: Amadio
authors.forename: Michela
authors.initials: M
authors.lastname: Giustizieri
authors.forename: Maria Letizia
authors.initials: ML
authors.lastname: Cucchiaroni
authors.forename: Alessandro
authors.initials: A
authors.lastname: Tozzi
authors.forename: Marie Claire
authors.initials: MC
authors.lastname: Velluz
authors.forename: Nicola Biagio
authors.initials: NB
authors.lastname: Mercuri
authors.forename: Cinzia
authors.initials: C
authors.lastname: Volonté
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D061988:Proteasome Inhibitors; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D051381:Rats; D058477:Receptors, Purinergic P2X3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D007976:Leupeptins; D061988:Proteasome Inhibitors; D058477:Receptors, Purinergic P2X3; D000255:Adenosine Triphosphate; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde