CELL-TO-CELL SIGNALLING MEDIATED VIA CO<SUB >2</SUB>: ACTIVITY DEPENDENT CO<SUB>2</SUB> PRODUCTION IN THE AXONAL NODE OPENS CX32 IN THE SCHWANN CELL PARANODE

Loss of function mutations of Cx32, which is expressed in Schwann cells, cause X-linked Charcot Marie Tooth disease, a slowly progressive peripheral neuropathy. Cx32 is thus essential for the maintenance of myelin. As Cx32 hemichannels are directly sensitive to CO₂, we have tested whether CO₂ produced in the axonal node, as a consequence of the energetic demands of action potential propagation, might gate Cx32 hemichannels. We have used a membrane impermeant fluorescent dye FITC, which can permeate Cx32 hemichannels, to demonstrate the opening of Cx32 in Schwann cells in response to an external CO₂ stimulus or during action potential propagation in the isolated nerve. Pharmacological blockade of APQ1 or allosteric enhancement of carbonic anhydrase activity greatly reduced Cx32 gating during action potential firing. By contrast, inhibition of carbonic anhydrase with acetazolamide greatly increased Cx32 gating. Schwann cell specific expression of GRABATP1.0 and GCaMP8 also showed CO2-dependent gating of Cx32 following action potential propagation. Cx32 gating was unaffected by the G-protein blocker GDPβS, indicating that it was not mediated by G protein coupled receptors. This CO₂-dependent opening of Cx32 also mediates an activity dependent Ca²⁺ influx into the paranode and, by increasing the leak current across the myelin sheath, slows the conduction velocity. Expression of dnCx32, a genetic tool to remove CO2 sensitivity from Cx32WT, prevented gating following action potentials. Our data demonstrate that CO₂ can act via connexins to mediate neuron-to-glia signalling and that CO₂ permeable aquaporins and carbonic anhydrase are key components of this signalling mechanism.