Cell-to-cell signalling mediated via CO 2 : activity dependent CO 2 production in the axonal node opens Cx32 in the Schwann cell paranode

Jack Butler, Lowell Mott, Amol Bhandare, Angus Brown, Nicholas Dale
2025 · doi:10.1101/2025.04.04.647196
preprint OA: gold CC-BY-4.0
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Abstract

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. During action potential propagation, Cx32 hemichannels in the Schwann cell paranode are thought to open and release ATP. As Cx32 hemichannels are directly sensitive to CO 2 , we have tested whether CO 2 produced in the axon, as a consequence of the energetic demands of action potential propagation, might gate Cx32 hemichannels. Using isolated sciatic nerve from the mouse, we have shown that the critical components required for intercellular CO 2 signalling are present (nodal mitochondria, the source of CO 2 ; a CO 2 -permeable aquaporin, AQP1; paranodal Cx32; and carbonic anhydrase). 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 CO 2 stimulus or during action potential propagation in the isolated nerve. Pharmacological blockade of AQP1 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. Cx32 gating was unabected by the G-protein blocker GDPβS, indicating that it was not mediated by G protein coupled receptors. By expressing a modified Cx32 subunit, Cx32 DN , that coassembles with Cx32 WT , we have shown that the activity dependent dye loading of Schwann cells depends upon CO 2 binding to Cx32. This CO 2 -dependent opening of Cx32 also mediates an activity dependent Ca 2+ influx into the paranode and, by increasing the leak current across the myelin sheath, slows the conduction velocity. Our data demonstrate that CO 2 can act via connexins to mediate neuron-to-glia signalling and that CO 2 permeable aquaporins and carbonic anhydrase are key components of this signalling mechanism.
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Abstract 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. During action potential propagation, Cx32 hemichannels in the Schwann cell paranode are thought to open and release ATP. As Cx32 hemichannels are directly sensitive to CO2, we have tested whether CO2 produced in the axon, as a consequence of the energetic demands of action potential propagation, might gate Cx32 hemichannels. Using isolated sciatic nerve from the mouse, we have shown that the critical components required for intercellular CO2 signalling are present (nodal mitochondria, the source of CO2; a CO2-permeable aquaporin, AQP1; paranodal Cx32; and carbonic anhydrase). 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 AQP1 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. Cx32 gating was unabected by the G-protein blocker GDPβS, indicating that it was not mediated by G protein coupled receptors. By expressing a modified Cx32 subunit, Cx32DN, that coassembles with Cx32WT, we have shown that the activity dependent dye loading of Schwann cells depends upon CO2 binding to Cx32. 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. 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. Competing Interest Statement The authors have declared no competing interest. Footnotes Revised in response to peer review from eLife. Added genetic manipulation of Cx32 sensitivity to CO2 to strengthen the evidence supporting CO2 as the messenger between axon and Schwann cell acting on Cx32; added evidence to link CO2 production to the Krebs cycle; added use of GCaMP8 expression in Schwann cells to show activity dependent Ca2+ influxes and their dependence on CO2 signalling. Data availability All data is included in the supplementary files for each figure.

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