Functional Requirement for a Highly Conserved Charged Residue at Position 75 in the Gap Junction Protein Connexin 32

Abrams, Charles K.; Islam, Mahee; Mahmoud, Rola; Kwon, Taekyung; Bargiello, Thaddeus A.; Freidin, Mona M.

doi:10.1074/jbc.m112.392670

Cited by 24 publications

(21 citation statements)

References 43 publications

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“…One other CNS mutant (R75W) previously studied by us32 is also included. In addition, we evaluated four mutants associated with typical CMT1X but lacking in CNS abnormalities; these patients had normal visual and brainstem auditory evoked potentials and no known episodes of acute, florid syndrome.…”

Section: Resultsmentioning

confidence: 99%

“…For heterotypic cell pairs, transfected cells were washed and cells expressing a pIRES2-EGFP construct expressing mutant Cx32 were mixed with cells expressing a pIRES2-DsRed construct expressing WT Cx32, in a 1:1 ratio. Coupling was assessed by dual whole-cell patch clamping of cell pairs 6–48 hours after re-plating as previously described32. Only cells clearly expressing the appropriate fluorescent proteins (thus indicating that they were transfected) were used for recording.…”

Section: Methodsmentioning

confidence: 99%

“…Until now only one of the clinically characterized mutations associated with florid CNS symptoms (R75W) has been examined electrophysiologically in mammalian expression systems, where the most reliable characterization can be made using dual whole-cell voltage clamp32. However, no systematic examination of the effects of clinically characterized mutations on their ability to form functional gap junctions has been undertaken.…”

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confidence: 99%

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Loss of Coupling Distinguishes GJB1 Mutations Associated with CNS Manifestations of CMT1X from Those Without CNS Manifestations

Abrams

Goman

Wong

et al. 2017

Sci Rep

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CMT1X, an X-linked inherited neuropathy, is caused by mutations in GJB1, which codes for Cx32, a gap junction protein expressed by Schwann cells and oligodendrocytes. Many GJB1 mutations cause central nervous system (CNS) abnormality in males, including stable subclinical signs and, less often, short-duration episodes characterized by motor difficulties and altered consciousness. However, some mutations have no apparent CNS effects. What distinguishes mutations with and without CNS manifestations has been unclear. Here we studied a total of 14 Cx32 mutations, 10 of which are associated with florid episodic CNS clinical syndromes in addition to peripheral neuropathy. The other 4 mutations exhibit neuropathy without clinical or subclinical CNS abnormalities. These “PNS-only” mutations (Y151C, V181M, R183C and L239I) form gap junction plaques and produce levels of junctional coupling similar to those for wild-type Cx32. In contrast, mutants with CNS manifestations (F51L, E102del, V139M, R142Q, R142W, R164W T55I, R164Q and C168Y) either form no morphological gap junction plaques or, if they do, produce little or no detectable junctional coupling. Thus, PNS and CNS abnormalities may involve different aspects of connexin function.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Loss of Coupling Distinguishes GJB1 Mutations Associated with CNS Manifestations of CMT1X from Those Without CNS Manifestations

Abrams

Goman

Wong

et al. 2017

Sci Rep

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show abstract

“…For heterotypic cell pairs, transfected cells were washed and cells expressing a pIRES2-EGFP construct were mixed with cells expressing a pIRES2-DsRed construct in a 1:1 ratio. Coupling was assessed by dual whole-cell patch clamping of cell pairs 6–48 hours after re-plating as previously described [2]. Recording solutions used were as follows (in mM): pipette solution, 145 CsCl 2 , 5 EGTA, 1.4 CaCl 2 , and 5.0 HEPES, pH 7.2; bath solution, 150 NaCl, 4 KCl, 1 MgCl 2 , 2 CaCl 2 , 5 dextrose, 2 pyruvate, and 10 HEPES, pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

A new mutation in GJC2 associated with subclinical leukodystrophy

et al. 2014

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Recessive mutations in GJC2, the gene encoding connexin 47 (Cx47), cause Pelizaeus-Merzbacher-like disease type 1 (PMLD1), a severe dysmyelinating disorder. One recessive mutation (p.Ile33Met) has been associated with a much milder phenotype - Hereditary spastic paraplegia type 44 (SPG44). Here we present evidence that a novel Arg98Leu mutation causes an even milder phenotype - a subclinical leukodystrophy. The Arg98Leu mutant forms gap junction plaques in HeLa cells comparable to wild-type Cx47, but electrical coupling was 20-fold lower in cell pairs expressing Arg98Leu than for cell pairs expressing wild-type Cx47. On the other hand, coupling between Cx47Arg98Leu and Cx43WT expressing cells did not show such reductions. Single channel conductance and normalized steady state junctional conductance-junctional voltage (Gj-Vj) relations differed only slightly from those for wild-type Cx47. Our data suggest that that the minimal phenotype in this patient results from a reduced efficiency of opening of Cx47 channels between oligodendrocyte and oligodendrocyte with preserved coupling between oligodendrocyte and astrocyte, and support a partial loss of function model for the mild Cx47 associated disease phenotypes.

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“…A large category of loss of function mutations result from large shifts in voltage-dependence, resulting in channel closure at physiological conditions where connexin channels would normally be fully open. 15 , 18 …”

Section: Introductionmentioning

confidence: 99%

Voltage Regulation of Connexin Channel Conductance

Bargiello

2015

Yonsei Med J

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Voltage is an important parameter that regulates the conductance of both intercellular and plasma membrane channels (undocked hemichannels) formed by the 21 members of the mammalian connexin gene family. Connexin channels display two forms of voltage-dependence, rectification of ionic currents and voltage-dependent gating. Ionic rectification results either from asymmetries in the distribution of fixed charges due to heterotypic pairing of different hemichannels, or by channel block, arising from differences in the concentrations of divalent cations on opposite sides of the junctional plaque. This rectification likely underpins the electrical rectification observed in some electrical synapses. Both intercellular and undocked hemichannels also display two distinct forms of voltage-dependent gating, termed Vj (fast)-gating and loop (slow)-gating. This review summarizes our current understanding of the molecular determinants and mechanisms underlying these conformational changes derived from experimental, molecular-genetic, structural, and computational approaches.

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Functional Requirement for a Highly Conserved Charged Residue at Position 75 in the Gap Junction Protein Connexin 32

Cited by 24 publications

References 43 publications

Loss of Coupling Distinguishes GJB1 Mutations Associated with CNS Manifestations of CMT1X from Those Without CNS Manifestations

Loss of Coupling Distinguishes GJB1 Mutations Associated with CNS Manifestations of CMT1X from Those Without CNS Manifestations

A new mutation in GJC2 associated with subclinical leukodystrophy

Voltage Regulation of Connexin Channel Conductance

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