Inhibitory modulation of distal C‐terminal on protein kinase C‐dependent phospho‐regulation of rat TRPV1 receptors

Abstract

The vanilloid receptor TRPV1, previously known as VR1, has been implicated in pain sensation under both physiological and pathological conditions. The channel is highly expressed in sensory ganglion neurones and is activated by a range of noxious stimuli including irritant chemicals, acids and heat. In order to understand the structural basis underlying this polymodal activation and the regulation by intracellular signalling pathways, we have investigated the functional roles of the cytoplasmic C-terminal of rat TRPV1. A mutant with the maximal truncation of the distal C-terminal encompassing the last 88 residues was constructed. Of interest, this mutant exhibited a Ca 2+ -dependent functional loss; it was irresponsive to capsaicin in the presence of extracellular Ca 2+ , but fully functional otherwise. Further studies of this construct revealed that extracellular Ca 2+ alone could activate the channel, and that the activation required protein kinase C (PKC) phosphorylation at S502, an event that was up-regulated by external Ca 2+ entry. We compared the truncation mutant with wild-type TRPV1 and demonstrated that it had a significantly increased sensitivity to PKC phosphorylation. These results suggest the distal C-terminal of TRPV1 can inhibit phosphorylation-induced potentiation of the wild-type channel. They also call into question some established functions of the distal C-terminal of TRPV1, including its roles in agonist binding and functional desensitization. We suggest that the functional loss of the truncation mutant, in the presence of extracellular Ca 2+ , was not due to disruption of agonist binding or gating, but rather to desensitization promoted by unstimulated extracellular Ca 2+ entry.

Modular thermal sensors in temperature-gated transient receptor potential (TRP) channels

“…The N terminus of the vanilloid receptors is relatively large in length, containing >300 residues or nearly half of the whole channel subunit, a significant portion of which is involved in formation of ankyrin repeats, a well-conserved structural motif in all vanilloid receptors and other subfamilies of TRP channels (1). The C terminus comprises 100-150 residues, of which the S6-linker region contains the TRP box, a signature sequence conserved among TRP channels, whereas a considerable portion of its distal end (approximately 88 residues in rTRPV1) is not required for temperature gating (20). Taking these features into consideration, we constructed chimeras involving exchanges of subregions in both termini ( Fig.…”

Uncoupling Proton Activation of Vanilloid Receptor TRPV1

“…Cells were maintained in DMEM plus 10% fetal bovine serum (HyClone Laboratories, Logan, UT) with 1% penicillin/streptomycin, incubated at 37°C in 5% CO 2 , and transfected at a confluence of ϳ80% using the standard calcium phosphate precipitation method as described previously (Liu et al, 2004). Either GFP (green fluorescent protein) or human CD8 lymphocyte antigen (0.5 g/0.2 ml) was cotransfected as surface markers.…”

“…The wild-type (WT) rat TRPV1 and TRPV2 cDNAs were generously provided by David Julius (University of California, San Francisco, San Francisco, CA) (Caterina et al, 1997(Caterina et al, , 1999. Mutations and chimeras were generated using the overlapextension PCR method as described previously (Liu et al, 2004). Fragments of residues that were exchanged between TRPV1 and TRPV2 were as follows (TRPV1/TRPV2): P456 -T476/ P415-L436 for the first extracellular loop between S1 and S2, S532-M541/M492-L501 for the second loop between S3 and S4, L585-L664/ L547-L627 for the pore loop, 601-611/E561-N671 for the turret, Y627-C634/Y590 -S597 for the pore helix, and D646 -V667/E609 -V631 for the S6-linker.…”

C-terminal Dimerization Activates the Nociceptive Transduction Channel Transient Receptor Potential Vanilloid 1

“…cys-cTRPV1-Truncation of the very end of rat TRPV1 produced sensitized mutant channels with lowered thresholds for temperature-and voltage-dependent activation, suggestive of a tonic inhibitory role of the distal region of the C terminus of TRPV1 in channel gating (22,23). An intersubunit disulfide bond within the segment just proximal to this "inhibitory" region might sensitize the full-length channel by relieving the inhibitory effect mediated by this distal C-terminal segment.…”