ANO1 (TMEM16A) is a Ca(2+)-activated Cl(−) channel (CaCC) expressed in peripheral somatosensory neurons that are activated by painful (noxious) stimuli. These neurons also express the Ca(2+)-permeable channel and noxious heat sensor TRPV1, which can activate ANO1. Here, we revealed an intricate mechanism of TRPV1-ANO1 channel coupling in rat dorsal root ganglion (DRG) neurons. Simultaneous optical monitoring of CaCC activity and Ca(2+) dynamics revealed that the TRPV1 ligand capsaicin activated CaCCs. However, depletion of endoplasmic reticulum (ER) Ca(2+) stores reduced capsaicin-induced Ca(2+) increases and CaCC activation, suggesting that ER Ca(2+) release contributed to TRPV1-induced CaCC activation. ER store depletion by plasma membrane-localized TRPV1 channels was demonstrated with an ER-localized Ca(2+) sensor in neurons exposed to a cell-impermeable TRPV1 ligand. Proximity ligation assays established that ANO1, TRPV1 and the IP(3) receptor IP(3)R1 were often found in close proximity to each other. Stochastic optical reconstruction microscopy (STORM) confirmed the close association between all three channels in DRG neurons. Together, our data reveal the existence of ANO1-containing multi-channel nanodomains in DRG neurons and suggest that coupling between TRPV1 and ANO1 requires ER Ca(2+) release, which may be necessary to enhance ANO1 activation.
