ANO1 (TMEM16A) is a Ca2+ activated Cl- channel (CaCC) with functions ranging from epithelial transport to sensory transmission.  ANO1 is expressed several tissue types, including epithelia, vasculature, as well as peripheral somatosensory neurons.  These neurons also express the Ca2+ permeable heat sensor TRPV1 and functional coupling between TRPV1 and ANO1 has been demonstrated. ANO1 is also robustly activated by the Ca2+ release from the endoplasmic reticulum (ER) through inositol trisphosphate receptors (IP3R). Interestingly, TRPV1 was shown to induce Ca2+ release from the ER by activating phospholipase C (PLC). Thus, here we investigated Ca2+ sources coupling TRPV1 and ANO1 activation; we hypothesised that TRPV1 may be able to activate ANO1 through IP3R Ca2+ release as well. To this end, we developed a multi-wavelength live cell imaging approach to simultaneously monitor CaCC activity and Ca2+ dynamics in the dorsal root ganglion (DRG) neurons. We observed that activation of TRPV1 with capsaicin was indeed able to induce CaCC. Furthermore, CaCC activity produced by capsaicin application was attenuated after depletion of the ER Ca2+ load, suggesting that ER Ca2+ release contributed to TRPV1-induced CaCC activation. To confirm that this effect was induced by plasmalemmal and not ER-localised TRPV1 channels, we used a cell impermeable TRPV1 activator, a derivative of double knot spider toxin, in combination with an ER-Ca2+ sensor to demonstrate that ER depletion only occurs when membrane-localised TRPV1 are activated. To understand if there was a structural arrangement of channels that allowed this coupling, we used in situ Proximity ligation assay (PLA) and stochastic optical reconstruction microscopy (STORM) to show that ANO1, TRPV1 and IP3R receptors were often found in close proximity in DRG neurons. In summary, our findings demonstrate that functional coupling between ANO1 and TRPV1 in sensory neurons is facilitated by Ca2+ release through IP3R with the channels found in a nanodomain structure to enable efficient ANO1 activation.

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