title: N-glycosylation determines ionic permeability and desensitization of the TRPV1 capsaicin receptor.
abstract: The balance of glycosylation and deglycosylation of ion channels can markedly influence their function and regulation. However, the functional importance of glycosylation of the TRPV1 receptor, a key sensor of pain-sensing nerves, is not well understood, and whether TRPV1 is glycosylated in neurons is unclear. We report that TRPV1 is N-glycosylated and that N-glycosylation is a major determinant of capsaicin-evoked desensitization and ionic permeability. Both N-glycosylated and unglycosylated TRPV1 was detected in extracts of peripheral sensory nerves by Western blotting. TRPV1 expressed in HEK-293 cells exhibited various degrees of glycosylation. A mutant of asparagine 604 (N604T) was not glycosylated but did not alter plasma membrane expression of TRPV1. Capsaicin-evoked increases in intracellular calcium ([Ca(2+)](i)) were sustained in wild-type TRPV1 HEK-293 cells but were rapidly desensitized in N604T TRPV1 cells. There was marked cell-to-cell variability in capsaicin responses and desensitization between individual cells expressing wild-type TRPV1 but highly uniform responses in cells expressing N604T TRPV1, consistent with variable levels of glycosylation of the wild-type channel. These differences were also apparent when wild-type or N604T TRPV1-GFP fusion proteins were expressed in neurons from trpv1(-/-) mice. Capsaicin evoked a marked, concentration-dependent increase in uptake of the large cationic dye YO-PRO-1 in cells expressing wild-type TRPV1, indicative of loss of ion selectivity, that was completely absent in cells expressing N604T TRPV1. Thus, TRPV1 is variably N-glycosylated and glycosylation is a key determinant of capsaicin regulation of TRPV1 desensitization and permeability. Our findings suggest that physiological or pathological alterations in TRPV1 glycosylation would affect TRPV1 function and pain transmission.
authors.affiliation: Department of Pharmacology, University of Melbourne, Parkville, Victoria, 3010, Australia.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Veldhuis
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Lew
authors.forename: Fe C
authors.initials: FC
authors.lastname: Abogadie
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Poole
authors.forename: Ernest A
authors.initials: EA
authors.lastname: Jennings
authors.forename: Jason J
authors.initials: JJ
authors.lastname: Ivanusic
authors.forename: Helge
authors.initials: H
authors.lastname: Eilers
authors.forename: Nigel W
authors.initials: NW
authors.lastname: Bunnett
authors.forename: Peter
authors.initials: P
authors.lastname: McIntyre
mesh_terms: D000818:Animals; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism; D004396:Coloring Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005822:Genetic Vectors; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007477:Ions; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009474:Neurons / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D010539:Permeability; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D050916:TRPV Cation Channels / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004396:Coloring Agents; D007477:Ions; D050916:TRPV Cation Channels; C494484:TRPV1 receptor; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase