Title : Biochemical characterization of the
vanilloid receptor 1 expressed in a dorsal root ganglia derived cell line
Abstract :
- The vanilloid receptor VR1 is an ion channel predominantly expressed by primary sensory neurons involved in nociception
- Here we describe its biochemical properties and assess the subcellular localization, the glycosylation state and the quaternary structure of VR1 expressed in HEK293 cells and in the DRG-derived cell line F-11 (N18TG2 mouse neuroblastoma x rat dorsal root ganglia, hybridoma)
- VR1 was found to be glycosylated in both cell types
- Of the five potential N-glycosylation sites , the predicted transient receptor potential channel-like transmembrane folding proposes N604 is localized extracellularly
- We used site-directed mutagenesis to mutate the Asn at position 604 to Thr
- This mutated VR1 was not glycosylated, confirming the extracellular location of N604 and its role as the exclusive site of glycosylation of the VR1 protein
- VR1 occured in high molecular mass complexes as assessed by blue native PAGE
- In the presence of limited amounts of SDS dimers , trimers and tetramers of VR1 were observed, consistent with the predicted tetrameric quaternary structure of the receptor
- Cross-linking with dimethyladipimidate yielded almost exclusively dimers
- Whereas VR1 localized both to the plasma membrane and to intracellular membranes in HEK293 cells, it localized predominantly to the plasma membrane in F-11 cells
- Using confocal laserscanning microscopy, we observed an enrichment of anti- VR1 immunoreactivity in neurite-like structures of F-11 cells
- In the light of conflicting literature data on biochemical characteristics of VR1 , our data suggest that dorsal root ganglion-derived F-11 cells provide a powerful experimental system for the study of VR1 biochemistry