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 language | English |
|---|---|
| Pages (from-to) | 634-643 |
| Number of pages | 10 |
| Journal | Glycobiology |
| Volume | 21 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - May 2011 |
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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 journal › Article
}
TY - JOUR
T1 - N-Glycans mutations rule oligomeric assembly and functional expression of P2X 3 receptor for extracellular ATP
AU - Vacca, Fabrizio
AU - D'Ambrosi, Nadia
AU - Nestola, Valeria
AU - Amadio, Susanna
AU - Giustizieri, Michela
AU - Cucchiaroni, Maria Letizia
AU - Tozzi, Alessandro
AU - Velluz, Marie Claire
AU - Mercuri, Nicola Biagio
AU - Volonté, Cinzia
PY - 2011/5
Y1 - 2011/5
N2 - 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.
AB - 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.
KW - αβmeATP
KW - inward currents
KW - oligomerization
KW - proteasome inhibition
KW - trafficking
UR - http://www.scopus.com/inward/record.url?scp=79953904606&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=79953904606&partnerID=8YFLogxK
U2 - 10.1093/glycob/cwq211
DO - 10.1093/glycob/cwq211
M3 - Article
VL - 21
SP - 634
EP - 643
JO - Glycobiology
JF - Glycobiology
SN - 0959-6658
IS - 5
ER -