Structures of wild-type and selected CMT1X mutant connexin 32 gap junction channels and hemichannels

Abstract

In myelinating Schwann cells, connection between myelin layers is mediated by gap junction channels (GJCs) formed by docked connexin 32 (Cx32) hemichannels (HCs). Mutations in Cx32 cause the X-linked Charcot-Marie-Tooth disease (CMT1X), a degenerative neuropathy without a cure. A molecular link between Cx32 dysfunction and CMT1X pathogenesis is still missing. Here, we describe the high-resolution cryo-electron microscopy (cryo-EM) structures of the Cx32 GJC and HC, along with two CMT1X-linked mutants, W3S and R22G. While the structures of wild-type and mutant GJCs are virtually identical, the HCs show a major difference: In the W3S and R22G mutant HCs, the amino-terminal gating helix partially occludes the pore, consistent with a diminished HC activity. Our results suggest that HC dysfunction may be involved in the pathogenesis of CMT1X.

Mechanism of connexin channel inhibition by mefloquine and 2-aminoethoxydiphenyl borate

“…The phenyl rings of 2APB are in contact with the hydrophobic side of the amphipathic NTH, in close proximity to the amino acids M1, G5, and L9. The lipid-2 density observed previously in the Cx32-apo structure 38 is located nearby and may contribute to 2APB binding. As we have imposed the D6 symmetry, we have modelled six 2APB molecules occupying the site A.…”

“…2c). The NTH, flexible in the apo-and MFQ-bound forms of the channel, changed to a conformation closely resembling the NTH conformation in the Cx32-apo HC structure 38 . An additional density between the NTH regions of the neighboring Cx32 monomers likely corresponds to 2APB.…”

“…1b-c, e-f). We used HEK293F cells which express Cx32 at the plasma membrane 38 and form functional gap junctions (Extended Data Fig. 1).…”

“…10a,c, Extended Data Table 1). The quality of the density maps was sufficient to resolve all of the features of the Cx32 GJC structure 38 , namely the transmembrane helices (TM1-4), extracellular loops 1 and 2 (ECL1-2) and lipid or detergent-like densities on the protein-lipid interface (Extended Data Fig. 8a, 9a).…”

“…The two drug binding sites (sites A and M) correspond to the previously described lipid binding sites that may bind either sterols or the acyl chains of phospholipid molecules 26,[38][39][40] . While binding of phospholipids to site A may stabilize the NTH in a closed conformation 39,40 , the functional properties of the site M have not been investigated prior to this study [38][39][40]42 . Our results suggest that drug binding to site A may affect NTH-mediated connexin pore gating (Fig.…”