Changes in Permeability Caused by Connexin 32 Mutations Underlie X-Linked Charcot-Marie-Tooth Disease

Oh, Seunghoon; Ri, Yi; Bennett, Michael V. L.; Trexler, E. Brady; Verselis, Vytas K.; Bargiello, Thaddeus A.

doi:10.1016/s0896-6273(00)80973-3

Cited by 239 publications

(246 citation statements)

References 48 publications

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“…This phenotype was first reported after removal of the conserved proline in M2 of Cx26 (Suchyna et al 1993) and later for the CMTX mutants M34T, V35M, and V38M in the first transmembrane domain of Cx32 (Oh et al 1997). In all cases, an accompanying shift in the conductance versus voltage relationship suggests that the closed state (or a residual low conductance state) is favored in the absence of an applied transjunctional field, a condition that favors the open state of wt channels (Suchyna et al 1993;Oh et al 1997;Skerrett et al 1999). Based on these observations, it appears as though the first transmembrane domain plays a critical role in the conformational changes that accompany Vj-dependent gating.…”

Section: Figurementioning

confidence: 75%

Site-Directed Mutagenesis Reveals Putative Regions of Protein Interaction within the Transmembrane Domains of Connexins

Toloue

Woolwine

Karcz

et al. 2008

Cell Communication & Adhesion

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Through cysteine-scanning mutagenesis, the authors have compared sites within the transmembrane domains of two connexins, one from the α-class (Cx50) and one from the β-class (Cx32), where amino acid substitution disrupts the function of gap junction channels. In Cx32, 11 sites resulted in no channel function, or an aberrant voltage gating phenotype referred to as "reverse gating," whereas in Cx50, 7 such sites were identified. In both connexins, the sites lie along specific faces of transmembrane helices, suggesting that these may be sites of transmembrane domain interactions. In Cx32, one broad face of the M1 transmembrane domain and a narrower, polar face of M3 were identified, including one site that was shown to come into close apposition with M4 in the closed state. In Cx50, the same face of M3 was identified, but sensitive sites in M1 differed from Cx32. Many fewer sites in M1 disrupted channel function in Cx50, and those that did were on a different helical face to the sensitive sites in Cx32. A more in depth study of two sites in M1 and M2 of Cx32 showed that side-chain length or branching are important for maintenance of normal channel behavior, consistent with this being a site of transmembrane domain interaction.

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Section: Figurementioning

confidence: 75%

Site-Directed Mutagenesis Reveals Putative Regions of Protein Interaction within the Transmembrane Domains of Connexins

Toloue

Woolwine

Karcz

et al. 2008

Cell Communication & Adhesion

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“…If the latter is the case, a taurine binding site could be composed of the CT domain (providing a loop-2-like motif) and the region of the transition between the NT domain and first transmembrane domain mentioned above (providing a loop-3-like motif). The NT-first transmembrane domain transition is thought to be at the mouth of the pore (50). The CT domain of Cx32 does not contain any of the motifs, which, in this hypothesis, is the reason it is unresponsive to aminosulfonate.…”

Section: Protonated Taurine Directly Modulates Connexinmentioning

confidence: 88%

Regulation of Connexin Channels by pH

Bevans¹,

Harris

1999

Journal of Biological Chemistry

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Protonated aminosulfonate compounds directly inhibit connexin channel activity. This was demonstrated by pH-dependent connexin channel activity in Good's pH buffers (MES (4-morpholineethanesulfonic acid), HEPES, and TAPS (3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino]-1-propanesulfonic acid)) that have an aminosulfonate moiety in common and by the absence of pH-dependent channel activity in pH buffers without an aminosulfonate moiety (maleate, Tris, and bicarbonate). The pH-activity relation was shifted according to the pK a of each aminosulfonate pH buffer. At constant pH, increased aminosulfonate concentration inhibited channel activity. Taurine, a ubiquitous cytoplasmic aminosulfonic acid, had the same effect at physiological concentrations. These data raise the possibility that effects on connexin channel activity previously attributed to protonation of connexin may be mediated instead by protonation of cytoplasmic regulators, such as taurine. Modulation by aminosulfonates is specific for heteromeric connexin channels containing connexin-26; it does not occur significantly for homomeric connexin-32 channels. The identification of taurine as a cytoplasmic compound that directly interacts with and modulates connexin channel activity is likely to facilitate understanding of cellular modulation of connexin channels and lead to the development of reagents for use in structure-function studies of connexin protein. Changes in intracellular pH (pH i )1 affect gap junction conductance between cells (1). The sensitivity of junctional conductance to changes in pH i varies with cell type and connexin isoform (2-5). Decrease of pH i from physiological levels typically produces a decrease in junctional conductance (6 -8) and in permeability to large tracers (9, 10). The decrease in junctional conductance is usually reversible with return of pH i to normal physiological values. The molecular mechanisms that underlie this modulation of connexin channel activity are unclear and may differ among connexin isoforms and cell types. It has been proposed that the modulation is due to direct protonation of connexin (8), changes in ionized calcium concentration (11), and activation of calmodulin (12-14). For connexin-43 and for connexin-32/connexin-38 chimerae, recent work strongly indicates a pH-dependent interaction between segments of the C-terminal domain and the single cytoplasmic loop that inhibits channel activity (3,(15)(16)(17)(18)(19)(20)(21).In this study, we set out to investigate modulation of connexin channel activity as a function of pH, using in a reconstituted system connexin channels immunoaffinity-purified from native tissues. Channel activity was monitored using transport-specific fractionation (TSF) of liposomes into which connexin channels were reconstituted.Channel activity in this system was affected by changes in pH. To our surprise, the changes in channel activity were accounted for by the direct action of the protonated form of the aminosulfonate compounds used as pH buffers, rather than of proton concen...

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“…This issue has been analyzed by expressing mutants in heterologous cells, in which many mutants do not form functional channels (Abrams et al, 2000) and typically do not traffic to the cell membrane (Yum et al, 2002). Other mutants form functional channels with altered biophysical characteristics; two of these (S26L and M34T) maintain electrical coupling but have an apparent reduction in their pore diameter, which may prevent the diffusion of small molecules (Oh et al, 1997). These studies also demonstrate that the nature of the mutation may be important, because the R15W and H94Q mutants form normal channels, whereas R15Q and .…”

Section: Genotype-phenotype Correlationsmentioning

confidence: 99%

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

Huang

Sirkowski²,

Stickney³

et al. 2005

J. Neurosci.

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Mutations in GJB1, the gene encoding the gap junction protein connexin32 (Cx32), cause the X-linked form of Charcot-MarieTooth disease, an inherited demyelinating neuropathy. The C terminus of human Cx32 contains a putative prenylation motif that is conserved in Cx32 orthologs. Using [ 3 H]mevalonolactone ([ 3 H]MVA) incorporation, we demonstrated that wild-type human connexin32 can be prenylated in COS7 cells, in contrast to disease-associated mutations that are predicted to disrupt the prenylation motif. We generated transgenic mice that express these mutants in myelinating Schwann cells. Male mice expressing a transgene were crossed with female Gjb1-null mice; the male offspring were all Gjb1-null, and one-half were transgene positive; in these mice, all Cx32 was derived from expression of the transgene. The mutant human protein was properly localized in myelinating Schwann cells in multiple transgenic lines and did not alter the localization of other components of paranodes and incisures. Finally, both the C280G and the S281x mutants appeared to "rescue" the phenotype of Gjb1-null mice, because transgene-positive male mice had significantly fewer abnormally myelinated axons than did their transgene-negative male littermates. These results indicate that Cx32 is prenylated, but that prenylation is not required for proper trafficking of Cx32 and perhaps not even for certain aspects of its function, in myelinating Schwann cells.

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Changes in Permeability Caused by Connexin 32 Mutations Underlie X-Linked Charcot-Marie-Tooth Disease

Cited by 239 publications

References 48 publications

Site-Directed Mutagenesis Reveals Putative Regions of Protein Interaction within the Transmembrane Domains of Connexins

Site-Directed Mutagenesis Reveals Putative Regions of Protein Interaction within the Transmembrane Domains of Connexins

Regulation of Connexin Channels by pH

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

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