Functional alterations in gap junction channels formed by mutant forms of connexin 32: evidence for loss of function as a pathogenic mechanism in the X-linked form of Charcot-Marie-Tooth disease

Abstract

CMTX, the X-linked form of Charcot-Marie-Tooth disease, is an inherited peripheral neuropathy arising in patients with mutations in the gene encoding the gap junction protein connexin 32 (Cx32). In this communication, we describe the expression levels and biophysical parameters of seven mutant forms of Cx32 associated with CMTX, when expressed in paired Xenopus oocytes. Paired oocytes expressing the R15Q and H94Q mutants show junctional conductances not statistically different from that determined for Cx32WT, though both show a trend toward reduced levels. The S85C and G12S mutants induce reduced levels of junctional conductance. Three other mutants (R15W, H94Y and V139M) induce no conductance above baseline when expressed in paired oocytes. Analysis of the conductance voltage relations for these mutants shows that the reduced levels of conductance are entirely (H94Y and V139M) or partly (S85C and R15W) explicable by a reduced open probability of the mutant hemichannels. The R15Q and H94Q mutations also show alterations in the conductance voltage relations that would be expected to minimally (H94Q) or moderately (R15Q) reduce the available gap junction communication pathway. The reduction in G12S induced conductance cannot be explained by alterations in hemichannel open probability and are more likely due to reduced junction formation. These results demonstrate that many CMTX mutations lead to loss of function of Cx32. For these mutations, the loss of function model is likely to explain the pathogenesis of CMTX.

Prenylation-Defective Human Connexin32 Mutants Are Normally Localized and Function Equivalently to Wild-Type Connexin32 in Myelinating Schwann Cells

“…It is interesting that some Cx32 mutants form functional gap junctions in transfected cells, including C280G and S281x (Castro et al, 1999;Abrams et al, 2001;Yum et al, 2002), yet cause CMT1X in humans. Perhaps these mutants do not traffic properly in myelinating Schwann cells, just as certain rhodopsin mutants do not traffic properly in rods (Sung et al, 1994), or perhaps they form channels with abnormal properties (Castro et al, 1999;Abrams et al, 2001;Liang et al, 2005).…”

“…Perhaps these mutants do not traffic properly in myelinating Schwann cells, just as certain rhodopsin mutants do not traffic properly in rods (Sung et al, 1994), or perhaps they form channels with abnormal properties (Castro et al, 1999;Abrams et al, 2001;Liang et al, 2005). To address this issue, we used a rat Mpz promoter to drive the expression of the human GJB1 gene (Fig.…”

“…Conversely, several disease-related mutants, including C280G and S281x, form fully functional channels (Castro et al, 1999;Abrams et al, 2001).…”

The Role of Gap Junctions in Charcot-Marie-Tooth Disease

“…Other mutants form functional channels with altered biophysical characteristics, such as reduced pore diameter that may prevent the diffusion of second messengers like IP 3 , cAMP, and Ca 2ϩ (Oh et al, 1997). The position of the Cx32 mutation alone does not necessarily predict the molecular and functional consequences, as the R15Q and H94Q mutants form normal functional channels, whereas R15W and H94Y do not (Abrams et al, 2001). When expressed in mammalian cells with more stringent protein trafficking requirements, Cx32 mutants are often retained intracellularly, even if they form rare GJ-like plaques (Omori et al, 1996;Yoshimura et al, 1998;Yum et al, 2002).…”

Voltage opens unopposed gap junction hemichannels formed by a connexin 32 mutant associated with X-linked Charcot-Marie-Tooth disease

“…Third, several other mutations lead to either no or very little detectable protein when expressed exogenously (12). Fourth, many mutants show alterations in their functional properties which would be predicted to lead to a complete or partial loss of coupling (13,14).…”