N-glycanase may play a role in pain transmission.

N-Glycosylation Determines Ionic Permeability and Desensitization of the TRPV1 Capsaicin Receptor*

June 22, 2012 The Journal of Biological Chemistry, 287, 21765-21772.

1. Nicholas A. Veldhuis^‡, 
2. Michael J. Lew^§, 
3. Fe C. Abogadie^‡, 
4. Daniel P. Poole^§,
5. Ernest A. Jennings^¶, 
6. Jason J. Ivanusic^§, 
7. Helge Eilers^‖, 
8. Nigel W. Bunnett** and
9. Peter McIntyre^‡,¹

+Author Affiliations

1. From the Departments of ^‡Pharmacology, and
2. ^§Anatomy and Neuroscience, University of Melbourne, Parkville, Victoria, 3010, Australia,
3. the ^¶School of Medicine and Dentistry, James Cook University, Cairns, QLD 4870, Australia,
4. the ^‖Department of Anesthesia and Perioperative Care, University of California, San Francisco, California 94143, and
5. the ^**Monash Institute of Pharmaceutical Sciences, Melbourne, Parkville, VIC 3052, Australia

1. ↵1 To whom correspondence should be addressed: Department of Pharmacology, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Grattan Street, Parkville, Victoria, Australia. Tel.: (+61)-3-8344-5745; Fax: (+61)-3-8344-5743; E-mail: pmci@unimelb.edu.au.

Capsule

Background: We studied effects of N-linked sugar residues on the sensory ion channel, TRPV1.

Results: Glycosylation of TRPV1 did not alter cell surface expression but was necessary for sustained cell calcium responses to allow uptake of YO-PRO-1 dye.

Conclusion: N-Glycosylation regulated inactivation and ion selectivity but not expression of TRPV1.

Significance: N-Glycosylation is a basic regulatory mechanism of TRPV1.

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 ([Ca²⁺]_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.