Regulation of Ca2+-activated Cl- channel ANO1 (TMEM16A) by different Ca2+ sources in sensory neurons

Proteins of anoctamin (TMEM16) family are the candidate subunits for Ca2+-activated Cl- channels (CaCC). In recent years, studies have shown that anoctamin-1 (TMEM16A or ANO1) plays important physiological roles in processes including epithelial fluid secretion, muscle contraction and olfactory transduction. How the Ca2+ regulates the activity of ANO1 in different tissue is still not clear.
In this study, I showed that the excitatory CaCC in nociceptors (small-diameter sensory neurons that are responsible for transmission of painful stimuli) was activated by the release of Ca2+ from the 1, 4, 5-trisphosphate (IP3)-sensitive intracellular stores in response to bradykinin (BK) or proteases (through protease activated receptor 2). Interestingly, while in the majority of nociceptors, CaCC was induced by Ca2+ release from the stores, only in a few neurons CaCC was activated by the Ca2+ influx through the voltage-gated Ca2+ channels (VGCC). Chelating intracellular Ca2+ with the slow Ca2+ buffer EGTA did not affect CaCC activation by protease activated receptor 2 (PAR2), while BAPTA abolished such activation, suggesting a close proximity of the Ca2+ release sites and CaCC. Membrane fractionation demonstrated that in the dorsal root ganglion (DRG), ANO1, bradykinin receptor 2 (B2R) or PAR2, were co-purified with lipid raft marker caveolin-1. Using various biochemical approaches I further demonstrated that ANO1 physically interacted with the IP3 receptor 1 (IP3R1) in DRG. Moreover, IP3R1, ANO1, B2R, and/or PAR2 were all assembled into functional signalling complexes and the plasma membrane components of the complex which contained ANO1 and GPCRs were tethered to the juxtamembrane regions of the endoplasmic reticulum. Disruption of the membrane microdomains by methyl-beta-cyclodextrin (MβCD) or competitive peptides partially restored coupling of CaCC to VGCC but disrupted coupling between B2R or PAR2 signaling and ANO1. Thus, such molecular complexes dichotomize different Ca2+ sources to provide ANO1-mediated excitation in response to specific ambient signals but protect the channels from global changes in intracellular Ca2+ and prevent sensory neurons from overexcitability.