X-linked Charcot Marie Tooth mutations alter CO₂ sensitivity of connexin32 hemichannels

Connexin32 (Cx32) is expressed in myelinating Schwann cells. It forms both reflexive gap junctions, to facilitate transfer of molecules from the outer to the inner myelin layers and hemichannels at the paranode to permit action potential-evoked release of ATP into the extracellular space. Loss of function mutations in Cx32 cause X-linked Charcot Marie Tooth disease (CMTX), a slowly developing peripheral neuropathy. The mechanistic links between Cx32 mutations and CMTX are not well understood. As Cx32 hemichannels can be opened by increases in PCO₂, we have examined whether CMTX mutations alter this CO₂ sensitivity. By using Ca²⁺ imaging, dye loading and genetically encoded ATP sensors to measure ATP release, we have found 5 CMTX mutations that abolish the CO₂ sensitivity of Cx32 hemichannels (A88D, 111-116 Del, C179Y, E102G, V139M). Others cause a partial loss (L56F, R220Stop, and R15W). Some CMTX mutations have no apparent effect on CO₂ sensitivity (R15Q, L9F, G12S, V13L, V84I, W133R). The mutation R15W alters multiple additional aspects of hemichannel function including Ca²⁺ and ATP permeability. The mutations that abolish CO₂ sensitivity are transdominant and abolish CO₂ sensitivity of co-expressed Cx32^WT. We have shown that Schwannoma RT4 D6P2T cells can release ATP in response to elevated PCO₂ via the opening of Cx32. This is consistent with the hypothesis that the CO₂ sensitivity of Cx32 may be important for maintenance of healthy myelin. Our data, showing a transdominant effect of certain CMTX mutations on CO₂ sensitivity, may need to be taken into account in any future gene therapies for this condition.