*3.2. TRPV1–ANO1 Interaction*

Approximately 80% of TRPV1-positive DRG neurons also express ANO1 by immunostaining [8,93]. To examine the function of this co-expression, we investigated whether these ion channels interact in

a setting of acute pain induced by capsaicin. We found that capsaicin-induced currents in isolated DRG neurons were suppressed by the ANO1 inhibitor, T16Ainh-A01 [94]. Although capsaicin-induced action potentials were also inhibited by T16Ainh-A01, inhibition was observed in response to second application of capsaicin (10 min after first capsaicin application). Some DRG neurons may not have exhibited second action potentials, yet there were DRG neurons that did show action potentials in the second application [93]. Another study showed that TRPV1 is desensitized by calmodulin binding. Moreover, TRPV1 function spontaneously and fully recovered after one hour, while desensitization was inhibited by TRPV1 phosphorylation [85]. Accordingly, the second response in some neurons is thought to depend on random phosphorylation levels in each neuron. Nonetheless, T16Ainh-A01 almost completely inhibits these second action potentials. These results suggest that chloride efflux elicited by ANO1 activation may accelerate depolarization to induce secondary action potentials, and that ANO1 inhibition may be effective at reducing pain sensation. In fact, capsaicin-induced pain-related behavior in mice is inhibited by T16Ainh-A01 [93]. Taken together, these findings indicate that the TRPV1–ANO1 interaction is critical for sensation of noxious stimuli.

ANO1 is also co-expressed with TRPV1 in TG neurons and is functionally involved in heat sensation [95,96]. In addition, TRPV1 and ANO1 expression levels are enhanced by estrogen in female rats [97]. Based on these observations, the TRPV1–ANO1 interaction may be a crucial target for pain therapies. According to a previous report, approximately 70% of ANO1-positive neurons do not colocalize with CGRP [8]. Although contribution of the TRPV1–ANO1 interaction in CGRP release is unknown, the TRPV1–ANO1 interaction may encompass the alternative side of the TRPV1–TMEM100–TRPA1 interaction system (Figure 1).

#### *3.3. Triple Conjugation of TRPV1, TRPA1, and TRPM3*

TRPM3 is a heat sensitive TRP channel that functionally couples with TRPV1 and TRPA1 [98]. Although TRPM3-deficient mice show a delayed tail flick at 57 ◦C, the effect of TRPM3 alone on heat sensation is unclear because tail flick behavior induced at 57 ◦C in TRPM3/TRPA1 double-deficient mice is no different to wild-type mice [4]. However, triple conjugation of TRPV1, TRPA1, and TRPM3 is important for detecting the noxious heat environment [4]. Withdrawal latency of TRPM3-deficient mice in the hot-plate test (50 ◦C) is the same as in wild-type mice [98]. Interestingly, this behavior disappears in TRPV1/TRPA1/TRPM3 triple-deficient mice, while the other responses to nociceptive stimuli are normal. Furthermore, wild-type and triple-deficient mice show a similar distribution on a gradient temperature plate (from 5 to 50 ◦C). In addition, CGRP-expressing DRG neurons are involved in heat sensation [62], and CGRP release from skin preparations is enhanced by the TRPM3 agonist, CIM0216, which is the same as for capsaicin treatment [99]. These findings indicate that the likely multiple function of TRPV1, TRPA1, and TRPM3 in peptidergic DRG neurons is to escape from a noxious heat environment.
