R. Jahnel et al., Biochemical characterization of the vanilloid receptor 1 expressed in a dorsal root ganglia derived cell line, EUR J BIOCH, 268(21), 2001, pp. 5489-5496
The vanilloid receptor VR1 is an ion channel predominantly expressed by pri
mary sensory neurons involved in nociception. Here we describe its biochemi
cal properties and assess the subcellular localization, the glycosylation s
tate and the quaternary structure of VR1 expressed in HEK293 cells and in t
he 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 recept
or potential channel-like transmembrane folding proposes N604 is localized
extracellularly. We used site-directed mutagenesis to mutate the Asn at pos
ition 604 to Thr. This mutated VR1 was not glycosylated, confirming the ext
racellular location of N604 and its role as the exclusive site of glycosyla
tion of the VR1 protein. VR1 occured in high molecular mass complexes as as
sessed by blue native PAGE. In the presence of limited amounts of SDS dimer
s, trimers and tetramers of VR1 were observed, consistent with the predicte
d tetrameric quaternary structure of the receptor. Cross-linking with dimet
hyladipimidate yielded almost exclusively dimers.
Whereas VR1 localized both to the plasma membrane and to intracellular memb
ranes in HEK293 cells, it localized predominantly to the plasma membrane in
F-11 cells. Using confocal laserscanning microscopy, we observed an enrich
ment 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 prov
ide a powerful experimental system for the study of VR1 biochemistry.