Researcher: Joe Lynch
Indication: Central Nervous System 
Background: Transient receptor potential type-1 cation channels are members of the six transmembrane (6TM) ion channel receptor family that includes most voltage-gated and several ligand-gated cation-selective ion channels. The TRPV family has six members (TRPV1-6) and four of these (TRPV1-4) are expressed in peripheral nociceptive sensory neurons. Although all four may have some potential as pain targets2, most attention has focussed on TRPV1 due to its sensitivity to a wide variety of noxious stimuli. Due to its permeability to Na and Ca, activation of TRPV1 induces membrane depolarisation and neuronal firing. This receptor is highly expressed in peripheral and central terminals of nociceptive dorsal root ganglion neurons3 mainly as a tetrameric homomer4, and its activators include heat (>43oC), capsaicin, low pH, and the endogenous ligand anandamide2,5. The synthetic analogue capsazepine is an antagonist of the TRPV1 agonist capasaicin. Several receptor-mediated second messenger pathways also converge to upregulate TRPV1 activity 2,5 Because these various noxious stimuli can induce activation either individually or cooperatively, TRPV1 is considered a polymodal integrator of pain stimuli. In support of a role in pain sensation, a wide variety of investigations employing models of inflammatory and neuropathic pain on both wildtype and TRPV1-/- mice have validated the contribution of TRPV1 to both types of pain2,5. These findings have prompted major commercial interest in developing TRPV1 antagonists as analgesics. One such compound, SB-705498, has entered Phase II clinical trials for migraine and several other molecules are in Phase I trials for various pain-related conditions6. Natural products that selectively inhibit TRPV1 could find application in the treatment of chronic inflammatory pain.
Theory: HEK293 cells transfected with the TRPV1 ion channel are loaded with a Ca2+ sensitive intracellular fluorophore (Fluo-4-AM). If TRPV1 is activated by an agonist, extracellular Ca2+ can flow through the open channel and stimulate intracellular fluorescence. Conversely, if the TRPV1 channel is exposed to the agonist capsaicin, in the presence of an antagonist from the test sample, the channel will remain closed and intracellular fluorescence will not occur. In this way the same TRPV1 transfected cell line and assay can be used to search for both agonists and antagonists.
Method: HEK293 cells are transfected with TRPV1 cDNA in DMEM in 6 cm dishes maintained at 37°C in a 5% CO2 atmosphere overnight, after which they are trypsinized and transferred into a 384-well plate (8000 cells/well in 40 uL) then washed with Ringer solution containing probenecid (to block efflux of intracellular dyes). Cells are then loaded with Fluo-4-AM by incubating at 37 °C for 1 hr in a Ringer-based solution containing Fluo-4-AM (10 uM), and again washed with Ringer. Test samples (~2 ug in 50 uL Ringer solution) are added to assay wells (to a final volume 70 uL), and the assays performed in duplicate.
References:
1 Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature. 1997. 389, 816-24.
2 Levine, J. D.; Alessandri-Haber, N. TRP channels: Targets for the relief of pain. Biochim. Biophys. Acta. 2007. 1772, 989-1003.
3 Sanchez, J. F., Krause, J. E., Cortright, D. N. The distribution and regulation of vanilloid receptor VR1 and VRi 5' splice variant RNA expression in rat. Neuroscience 2001. 107, 373-81.
4 Garcia-Sanz, N., Fernandez-Carvajal, A., Morenilla-Palao, C., Planells-Cases, R., Fajardo-Sanchez, E., Fernandez-Ballester, G., Ferrer-Montiel, A. Identification of a tetrametrization domain in the C terminus of the vanilloid receptor. J. Neurosci. 2004. 24, 5307-14.
5 Szallasi, A., Cruz, F., Geppetti, P. TRPV1: a therapeutic target for novel analgesic drugs? Trends Mol. Med. 2006. 12, 545-54.
6 Westaway, S. M. The potential of transient receptor potential vanilloid type 1 channel modulators for the treatment of pain. J. Med. Chem. 2007. 50, 2589-96.
A first fluorescence image of each well is taken before addition of the test sample to confirm that the cells are intact and the TRPV1 channels are closed (dim intracellular fluorescence). Shortly (30 sec) after addition of the test sample a second image is taken to establish if an agonist has opened the TRPV1 channel (ie stimulated intracellular fluorescence). A third image is taken after the addition of capsaicin to confirm that unopened TPRV1 channels are still functional (ie induce intracellular fluorescence). In the event that the test sample contained an antagonist this should be evident from a failure of the capsaicin to open the TRPV1 channel (ie no fluorescence even in the third image). All images are segmented and analysed automatically (λexc= 488 nm, λem= 520 nm; 300 to 1000 cells/image, which equates to 600 to 2000 cells per test sample), measuring and comparing the fluorescence in each cell before and after addition of the test sample and capsaicin control.
Full Title: TRPV1 Modulators