Supercooling agent icilin blocks a warmth-sensing ion channel TRPV3

Abstract

Transient receptor potential vanilloid subtype 3 (TRPV3) is a thermosensitive ion channel expressed in a variety of neural cells and in keratinocytes. It is activated by warmth (33-39°C), and its responsiveness is dramatically increased at nociceptive temperatures greater than 40°C. Monoterpenoids and 2-APB are chemical activators of TRPV3 channels. We found that Icilin, a known cooling substance and putative ligand of TRPM8, reversibly inhibits TRPV3 activity at nanomolar concentrations in expression systems like Xenopus laeves oocytes, HEK-293 cells, and in cultured human keratinocytes. Our data show that icilin's antagonistic effects for the warm-sensitive TRPV3 ion channel occurs at very low concentrations. Therefore, the cooling effect evoked by icilin may at least in part be due to TRPV3 inhibition in addition to TRPM8 potentiation. Blockade of TRPV3 activity by icilin at such low concentrations might have important implications for overall cooling sensations detected by keratinocytes and free nerve endings in skin. We hypothesize that blockage of TRPV3 might be a signal for cool-sensing systems (like TRPM8) to beat up the basal activity resulting in increased cold perception when warmth sensors (like TRPV3) are shut off.

Figure 1

Block of mTRPV3 activity by icilin in oocytes. (a) A representative trace showing camphor, menthol, and 2-APB activation of mTRPV3, whereas icilin could not activate in Ca²⁺-containing SES. (b) A representative trace showing block of mTRPV3 activation by icilin in Ca²⁺-free SES. (c) Block of camphor-induced mTRPV3 currents by icilin. (d) Dose-dependent block of mTRPV3 by icilin in Ca²⁺-containing SES. (e) Inhibition curve for icilin's IC₅₀ values. (f) Reversibility of inhibition of mTRPV3 by increasing 2-APB concentrations. (n = 6 in each case).

Figure 2

(a) Representative recordings showing an inhibition of mTRPV3 activity at different holding potentials. The upper and lower part of the trace shows a block of 2-APB-induced mTRPV3 activity at + or −40 mV holding potentials, respectively. (b) Quantification of the data from six experiments as explained above. (c) The upper and lower part of the trace shows a block of camphor induced mTRPV3 activity at + or −40 mV holding potentials, respectively. (d) Quantification of the data from six experiments as explained above. All values are expressed as mean ± SEM.

Figure 3

(a) A representative recording from patch-clamp experiment showing camphor-induced mTRPV3 activity inhibited by 100 nM icilin (61% inhibition; n = 6). HEK293 cells expressing mTRPV3 were held at –40 mV holding potential at ~33°C. (b) A representative recording from patch-clamp experiment showing 2-APB-induced mTRPV3 activity inhibited by 100 nM icilin (68% inhibition; n = 7) under similar conditions as above. (c) A representative recording from patch-clamp experiment showing icilin-dependent (100 nM) inhibition of mTRPV3 activity induced by both camphor and 2-APB (74% inhibition; n = 7) under similar conditions as above.

Figure 4

Block of TRPV3 in keratinocytes. Representative ratio-fluoremetric recordings of fura-2-loaded human primary keratinocytes are shown. Changes in cytosolic Ca²⁺ levels upon stimulation are depicted as fluorescence ratio (f ₃₄₀/f _380 nm) and displayed as a function of time. Transient stimulation by either 300 μM 2-APB (b) or 6 mM camphor (c) results in a Ca²⁺-influx. In contrast, stimulation by 10 μM icilin had no effect on the intracellular calcium concentration (a). The physiological state of the cells was controlled by application of 100 μM ATP at the end of the experiment. Upon coapplication of icilin induced Ca²⁺-signals by 2-APB (b) or camphor. (c) were dramatically reduced, respectively. Bars above denote the application points of the stimuli (black: stimulus; grey: coapplication of icilin).

Figure 5

Original trace showing block of human TRPV3 by icilin. Since lower concentrations of icilin were not effective in case of hTRPV3 so higher doses were tested. 0.3 mM of icilin blocked ~50% of response induced by 10 mM 2-APB. The oocyte was preincubated in icilin before application of 2-APB. The experiment was carried out in Ca²⁺-free SES.