Neuropathic pain — the role of anoctamin-6
Neuropathic pain is a result of microgliosis in the spinal cord in response to peripheral nerve injury that is poorly understood and has limited treatment options. Microgliosis is a process of microglial cells' activation in the spinal cord to remove damaged peripheral sensory nerves while activating pain sensation. The EU-funded 'Microglia and neuropathic pain: The role of calcium activated chloride channels' (CACCINNP) project investigated the molecular mechanisms involved in microglial cell activation that are associated with neuropathic pain. In particular, a membrane protein called anoctamin-6, associated with chloride (Cl-) transport regulation and membrane asymmetry, was studied. Researchers utilised a novel genetically encoded indicator called Cl-Sensor and having a genetic mutation that increases sensitivity to Cl-. Two mouse lines were created that expressed this Cl- sensor to functionally characterise intra–cellular Cl- changes in nerve cells using ratiometric tools. Anoctamin-6's role as a Cl- channel was confirmed in a heterologous system transiently transfected with anoctamin-6 cDNA. Fluourescent-activated cell sorting, site-directed mutagenesis and time-lapse microscopy were used to reveal its role in calcium-dependent scrambling of phospholipids such as phosphatidylserine (PS). PS dysfunction was assessed using conditional Anoctamin-6 knock-out (KO) mice. Diminished phagocytosis and scramblase activity were seen in the mice. Mutations in these KO mice resembled the rare congenital bleeding disorder called Scott Syndrome seen in some patients. In a mouse model for neuropathic pain, an increase in microgliosis was seen in neuropathic cells. Further studies should further elucidate the role of this protein in neuropathic pain. Tools developed by the CACCINNP project have enabled non-invasive monitoring of intra–cellular Cl- changes. Results of the CACCINNP project could provide novel targets for neuropathic pain therapy, providing relief to patients.