Functional Coupling in Nociceptive Sensory Neurons Between Ip3 Receptors and the Calcium-Activated Ano1(TMEM16A) Chloride Channel

Abstract

Ca2+-activated Cl- channels (CaCCs) are an important group of ion channels with diverse physiological roles whose molecular identity long time remained enigmatic. Members of the anoctamin (ANO) or TMEM16 family of proteins were recently identified as likely CaCC candidates. Thus, ANO1 (TMEM16A) mediates CaCC currents in epithelial and smooth muscle cells and in damage-sensing (nociceptive) sensory neurons. We report that ANO1 channels in small neurons from dorsal root ganglia (DRG) are preferentially activated by particular pools of intracellular Ca2+. As demonstrated by patch-clamp and iodide imaging experiments, these ANO1 channels can be selectively activated by the G protein-coupled receptor (GPCR)-induced release of Ca2+ from intracellular stores, but not by Ca2+ influx through voltage-gated Ca2+ channels. Co-immunoprecipitation experiments and proximity ligation assay (PLA) suggested that this ability to discriminate between Ca2+ pools was achieved by the tethering of ANO1-containing plasma membrane domains to juxtamembrane regions of the endoplasmic reticulum. The ANO1-containing plasma membrane microdomains were assembled within lipid rafts and also contained GPCRs such as bradykinin receptor-2 and protease-activated receptor-2. As revealed by GST pull-down and peptide competition electrophysiology, interaction of the C-terminus and the first intracellular loop of ANO1 with IP3R1 (inositol 1,4,5-trisphosphate receptor 1) contributed to the tethering. Disruption of membrane microdomains by cholesterol extraction blocked the ANO1 and IP3R1 interaction and resulted in the loss of coupling between GPCR signaling and ANO1. The junctional signaling complex enabled ANO1-mediated excitation in response to specific Ca2+signals rather than to global changes in intracellular Ca2+.