Nanodomain Calcium Signals Couple Activation of TRPV1 and ANO1 Sensory Ion Channels

Abstract

ANO1 (TMEM16A) is a Ca2+ activated Cl- channel (CaCC) expressed in peripheral somatosensory neurons responding to noxious stimuli; ANO1 amplifies nociceptive signals generated by Ca2+ permeable channels/receptors. Previous work in our lab has revealed ANO1 activation is coupled to Ca2+ release through IP3 receptors (IP3R); this is facilitated by these proteins assembling as multiprotein complexes at endoplasmic reticulum-plasma membrane (ER-PM) junctions meaning close proximity between ANO1 and IP3R allows the poor Ca2+ sensitivity of ANO1 to be overcome. Interestingly, ANO1 is a heat sensor with heat thresholds similar to that of TRPV1, the classical heat sensor of the body; previous studies have suggested coupling between these channels. We used live confocal imaging of Cl- channel activity, proximity ligation assay (PLA) and super-resolution microscopy to further probe the functional interaction between TRPV1 and ANO1 in dorsal root ganglion (DRG) neurons. Simultaneous imaging of Cl- channel activity with a halide sensitive EYFP mutant (H148Q/I152L), and Ca2+ dynamics with fura-2 revealed that capsaicin, a TRPV1 agonist, robustly activated CaCC. Intriguingly, ER-Ca2+ depletion severely attenuated CaCC activation suggesting that a major component of TRPV1-induced CaCC activation is through ER-Ca2+ release. Imaging ER-Ca2+ content with the ER-localised Ca2+ indicator CEPIA confirmed capsaicin-mediated Ca2+ release from the ER. Inhibition of plasma membrane localised TRPV1 with the cell-impermeable inhibitor, ruthenium red, abolished capsaicin-induced Ca2+ transients, suggesting that capsaicin-induced ER-Ca2+ release is not mediated by ER-localised TRPV1 channels. PLA revealed close proximity (<40nm) between ANO1, TRPV1 and IP3R1 in DRG neurons. Finally, Stochastic Optical Reconstruction (STORM) microscopy confirmed the close association between these proteins- 40% of clusters detected consisted of ANO1, TRPV1 and IP3R1. Taken together, our data indicates the existence of TRPV1:ANO1:IP3R1 multichannel complexes in DRG neurons and suggests involvement of ER-Ca2+ in ANO1-TRPV1 coupling.