Transient Receptor Potential Melastatin 8 (TRPM8) interacts with co-expressed Transient Receptor Potential Vanilloid 1 (TRPV1) and Ankyrin 1 (TRPA1)

Laura Sadofsky, Christopher Crow, Yvette Hayman, Alyn Morice. University of Hull, Hull, UK

Functional transient receptor potential (TRP) channels are thought to be tetramers, possibly either homotetramers or heterotetramers. In vivo, the nociceptors transient receptor potential ankyrin 1 and vanilloid 1 (TRPA1 and TRPV1 respectively) are expressed in the same sensory neurons and it has been suggested that these two channels can interact (Story et al, 2003). Transient receptor potential melastatin 8 (TRPM8) is however usually expressed on a distinct subset of neurons. The three channels have been extensively characterised independently, however to create a naturalistic model in order to understand how this nociceptor system functions and the channels interact we cloned the three channels and created a cell line permanently expressing both TRPA1 and TRPV1 (TRPA1/V1-HEK) as well as a separate cell line expressing TRPM8 (TRPM8-HEK). In order to characterise these cell lines we performed calcium signalling experiments with known TRPA1, TRPV1 and TRPM8 agonists. The cells were loaded with Fluo-3AM and experiments were performed using a fluorospectrometer. The TRPA1/V1-HEK cell line responded to both cinnamaldehyde and capsaicin in concentration dependent manners from 3 µM to 3 mM and from 1 nM to 300 nM respectively, with both agonists evoking maximum responses of 58% of calcium ionophore (maximum response). These results were similar to the responses seen for HEK293 cells permanently expressing TRPV1 or TRPA1 independently. Pre-incubation of cells with increasing concentrations of capsaicin followed by challenge with the EC50 concentration of cinnamaldehyde or vice versa caused a concentration dependent decrease in response to the second agonist, possibly through desensitisation. TRPM8-HEK cells responded well to menthol and WS-12. No change in signalling to menthol or cinnamaldehyde was observed with co-culture of TRPA1/V1 with TRPM8-HEK cells for 24 hrs prior to calcium signalling experiments, however when TRPM8-HEK cells were activated with WS-12 before adding to the TRPA1/V1-HEK cells, a reduction of 20% of the maximum response (at 300 µM) was seen with the TRPA1/V1-HEK cinnamaldehyde concentration effect curve. Interestingly, addition of the supernatant only from the WS-12 activated TRPM8-HEK cells to the TRPA1/V1-HEK cells prior to signalling also caused a 20% reduction in the cinnamaldehyde concentration effect curve maximum response (at 300 µM). The results suggest that TRPA1/V1-HEK cells are inhibited by a diffusible mediator/mediators from the agonist activated TRPM8-HEK cells.

Story et al (2003) Cell 112: 819-829