Cx32 hemichannel opening by cytosolic Ca2+ is inhibited by the R220X mutation that causes Charcot-Marie-Tooth disease

Hum Mol Genet. 2018 Jan 1;27(1):80-94. doi: 10.1093/hmg/ddx386.


Mutations of the GJB1 gene encoding connexin 32 (Cx32) cause the X-linked form of Charcot-Marie-Tooth disease (CMTX1), a demyelinating peripheral neuropathy for which there is no cure. A growing body of evidence indicates that ATP release through Cx32 hemichannels in Schwann cells could be critical for nerve myelination, but it is unknown if CMTX1 mutations alter the cytosolic Ca2+-dependent gating mechanism that controls Cx32 hemichannel opening and ATP release. The current study uncovered that loss of the C-terminus in Cx32 (R220X mutation), which causes a severe CMTX1 phenotype, inhibits hemichannel opening during a canonical IP3-mediated increase in cytosolic Ca2+ in HeLa cells. Interestingly, the gating function of R220X hemichannels was completely restored by both the intracellular and extracellular application of a peptide that mimics the Cx32 cytoplasmic loop. All-atom molecular dynamics simulations suggest that loss of the C-terminus in the mutant hemichannel triggers abnormal fluctuations of the cytoplasmic loop which are prevented by binding to the mimetic peptide. Experiments that stimulated R220X hemichannel opening by cell depolarization displayed reduced voltage sensitivity with respect to wild-type hemichannels which was explained by loss of subconductance states at the single channel level. Finally, experiments of intercellular diffusion mediated by wild-type or R220X gap junction channels revealed similar unitary permeabilities to ions, signalling molecules (cAMP) or larger solutes (Lucifer yellow). Taken together, our findings support the hypothesis that paracrine signalling alteration due to Cx32 hemichannel dysfunction underlies CMTX1 pathogenesis and suggest a candidate molecule for novel studies investigating a therapeutic approach.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Calcium / metabolism*
  • Calcium Channels / genetics
  • Calcium Channels / metabolism*
  • Charcot-Marie-Tooth Disease / genetics
  • Charcot-Marie-Tooth Disease / metabolism*
  • Connexins / antagonists & inhibitors
  • Connexins / chemistry
  • Connexins / genetics*
  • Connexins / metabolism*
  • Cytosol / metabolism
  • Gap Junctions / genetics
  • Gap Junctions / metabolism
  • HeLa Cells
  • Humans
  • Ion Channel Gating / physiology
  • Membrane Potentials / physiology
  • Models, Molecular
  • Mutation*
  • Patch-Clamp Techniques
  • Schwann Cells / metabolism
  • Transfection


  • Calcium Channels
  • Connexins
  • connexin 32
  • Adenosine Triphosphate
  • Calcium