Three putative N-glycosylation sites within the murine 5-HT3A receptor sequence affect plasma membrane targeting, ligand binding, and calcium influx in heterologous mammalian cells.
The serotonin type 3(A) receptor (5-HT3(A)R) is a ligand-gated ion channel (LGIC) that modulates a diverse set of cognitive and physiological functions. The 5-HT3(A)R, as with other LGICs, is a pentameric ion channel comprising five glycoprotein subunits. Although the N-terminal of the 5-HT3(A)R contains three putative N-linked glycosylation sites, the importance of each glycosylation site has not yet been established. To address this question, we used tunicamycin treatment and site-directed mutagenesis to inhibit selectively N-linked glycosylation at each site and then examined the effects of these treatments on receptor expression and function in transiently transfected heterologous cells. We show that the murine 5-HT3(A)R is glycosylated and that each N-linked glycosylation site plays a role in receptor regulation. Our findings suggest that N109 is necessary for receptor assembly, whereas N174 and N190 are important for plasma membrane targeting and ligand binding. Furthermore, we demonstrate that each site is necessary for 5-HT3(A)R-mediated Ca(2+) influx. We conclude that N-glycosylation is a critical step in the maturation, trafficking, and function of the murine 5-HT3(A)R.
Quirk
Phillip L
PL
Department of Pharmacology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106-4965, USA.
Rao
Suma
S
Roth
Bryan L
BL
Siegel
Ruth E
RE
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D006031:Glycosylation; D008024:Ligands; D051379:Mice; D016297:Mutagenesis, Site-Directed; D021381:Protein Transport / Q000235:genetics; D044406:Receptors, Serotonin, 5-HT3 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism; D009435:Synaptic Transmission / Q000235:genetics; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D008024:Ligands; D044406:Receptors, Serotonin, 5-HT3; D014415:Tunicamycin; D012701:Serotonin; D002118:Calcium