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

Fabrizio Vacca, Nadia D'Ambrosi, Valeria Nestola, Susanna Amadio, Michela Giustizieri, Maria Letizia Cucchiaroni, Alessandro Tozzi, Marie Claire Velluz, Nicola Biagio Mercuri, Cinzia Volonté

Research output: Contribution to journalArticle

11 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)634-643
Number of pages10
JournalGlycobiology
Volume21
Issue number5
DOIs
Publication statusPublished - May 2011

Fingerprint

Glycosylation
Purinergic P2 Receptors
Polysaccharides
Purinergic P2X3 Receptors
Adenosine Triphosphate
Mutation
Association reactions
Mutagenesis
Proteins
Protein Stability
HEK293 Cells
Protein Transport
Cell membranes
Site-Directed Mutagenesis
Ion Channels
Membrane Proteins
Cell Membrane
Ligands
Membranes

Keywords

  • αβmeATP
  • inward currents
  • oligomerization
  • proteasome inhibition
  • trafficking

ASJC Scopus subject areas

  • Biochemistry

Cite this

N-Glycans mutations rule oligomeric assembly and functional expression of P2X 3 receptor for extracellular ATP. / Vacca, Fabrizio; D'Ambrosi, Nadia; Nestola, Valeria; Amadio, Susanna; Giustizieri, Michela; Cucchiaroni, Maria Letizia; Tozzi, Alessandro; Velluz, Marie Claire; Mercuri, Nicola Biagio; Volonté, Cinzia.

In: Glycobiology, Vol. 21, No. 5, 05.2011, p. 634-643.

Research output: Contribution to journalArticle

Vacca, Fabrizio ; D'Ambrosi, Nadia ; Nestola, Valeria ; Amadio, Susanna ; Giustizieri, Michela ; Cucchiaroni, Maria Letizia ; Tozzi, Alessandro ; Velluz, Marie Claire ; Mercuri, Nicola Biagio ; Volonté, Cinzia. / N-Glycans mutations rule oligomeric assembly and functional expression of P2X 3 receptor for extracellular ATP. In: Glycobiology. 2011 ; Vol. 21, No. 5. pp. 634-643.
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