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

Abstract

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.

Cues to Opening Mechanisms From in Silico Electric Field Excitation of Cx26 Hemichannel and in Vitro Mutagenesis Studies in HeLa Transfectans

“…The relevance of connexins and their tightly regulated function is highlighted by their implication in pathological states of completely different character, such as cancer (Tsai et al, 2018 ), inflammation (Li et al, 2018 ), and neurodegenerative diseases (Belousov et al, 2018 ). For this reason, modulation of connexin hemichannels is becoming increasingly interesting for the treatment of several diseases including Alzheimer Disease (Yi et al, 2017 ), skin disorders, or X-linked Charcot Marie Tooth disease (Sáez and Leybaert, 2014 ; Carrer et al, 2017 ; Xu et al, 2017 ).…”

What’s the Function of Connexin 32 in the Peripheral Nervous System?

“…In 1997, Oh et al (Oh et al, 1997 ) hypothesized that the primary defect underlying CMT1X neuropathy in the presence of Cx32 mutants forming electrically conductive channels is the lower permeability of GJ channels to cAMP, which is involved in myelin homeostasis in SCs (LeBlanc et al, 1992 ). We have recently demonstrated that this theory is unlikely, at least with regard to the most studied CMT1X mutant (R220X; Carrer et al, 2018 ). Indeed, lack of Cx32 GJs in myelinating SCs does not appear to cause a slower radial diffusion of low molecular weight dyes along the myelin sheath of Cx32-null mouse with respect to the WT due to the presence of other connexins forming GJ channels (Balice-Gordon et al, 1998 ).…”

“…Most of these studies were limited to testing only GJ channel electrical conductance, so minimal information is available about specific permeability to important molecules up to 1 kDa (e.g., second messengers), which could explain why some mutants appear as “functional” with respect to the WT. Limited information is also available about gating/permeability dysfunction of mutant Cx32 hemichannels, analyzed in the following studies: S26L (Mones et al, 2014 ), S85C (Abrams et al, 2002 ), D178Y (Gómez-Hernández et al, 2003 ), E208K-Y211X-R215X-R215W-R215Q-C217X (Castro et al, 1999 ), R220X (Castro et al, 1999 ; Carrer et al, 2018 ), F235C (Liang et al, 2005 ), R238H-R265X-C280G-S281X (Castro et al, 1999 ).…”

“…Interaction between Cx32 hemichannels and mitochondria may play a role in cell bioenergetics as found in liver of Cx32-null mice (Fowler et al, 2013 ). (B) Cx32 mutations belonging to classes 3-4-5 mentioned in the text are represented as colored circles (black-red-azure, respectively) associated to the correspondent WT amino acid (white circle), where the topology of Cx32 domains is derived from the all-atom model of Cx32 connexon in Carrer et al ( 2018 ).…”

The two faces of pannexins: new roles in inflammation and repair

“… 135 Numerous reports since then have demonstrated the sensitivity of connexin HCs to extracellular Ca 2+ , which are believed to keep connexin HCs in a closed state at physiological Ca 2+ levels. 136 – 138 In contrast, Panx HCs are not gated by external Ca 2+ , 139 and the mechanical sensitivity of pannexin HCs was not noted until 2004, when single-channel currents were elicited by changes in pressure imposed pneumatically upon membrane patches of Xenopus oocytes expressing Panx1. 140 Since then, mechanosensitive purinergic signaling pathways, including pannexin-mediated ATP release, have been demonstrated in many cell types in response to mechanical stimuli.…”