Understudied chloride channels of the rat Dorsal Root Ganglia – ionotropic glycine receptors and Anoctamin 6

Current analgesics are often inefficacious or have serious side effects, as with centrally acting opioids, leaving many patients suffering and preventing their engagement in day-to-day life. Recent evidence suggests a GABA-mediated chloride conductance acts to inhibit nociceptive signals at the Dorsal Root Ganglia (DRG), the location of primary sensory neurone somata. This peripheral chloride conductance represents a novel therapeutic target for analgesia, reducing the risks associated with targeting the central nervous system. Here, hitherto under-investigated chloride channels which could potentially contribute to peripheral inhibition of nociception were examined and the associated consequences for nociceptive signalling discussed. Investigation of the ionotropic glycine receptor (iGlyR) in rat DRG revealed robust expression of only the accessory β subunit, but limited expression of the pore-forming α subunits. This was further supported by a lack of functional evidence of glycine-induced electrogenic responses in DRG neurones. The proposed calcium-activated chloride channel (CaCC) and phospholipid scramblase (PLS) Anoctamin 6 (ANO6, TMEM16F) was found expressed in DRG, and multiple live-cell fluorescent imaging approaches confirmed that ANO6 contributes to calcium-induced phospholipid scrambling in DRG neurones, and to the movement of both anions and cations across neuronal membranes. Electrophysiological recordings were unable to provide evidence that ANO6 is a bonafide ion channel, suggesting ANO6-mediated ion transport is a consequence of PLS. Nanoparticle tracking analysis (NTA) was employed to investigate whether ANO6 PLS could be linked to exosome release and exosomal communication within the DRG. Neither a pan-anoctamin activator or partial ANO6 knockdown significantly affected the exosome population isolated from DRG, but further investigation is warranted before a contribution of ANO6 PLS to exosome release can be completely ruled out. In sum, ANO6 has been identified as underlying a proportion of PLS in DRG, with potential implications for intraganglionic communication and the modulation of nociceptive signalling in the peripheral nervous system.