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The connexin46 mutant, Cx46T19M, causes loss of gap junction function and alters hemi-channel gating.

Tong JJ, Minogue PJ, Kobeszko M, Beyer EC, Berthoud VM, Ebihara L - J. Membr. Biol. (2014)

Bottom Line: In transfected HeLa cells, T19M was largely localized within the cytoplasm, with drastically reduced formation of gap junction plaques.When incubated in medium containing physiological concentrations of divalent cations, T19M-expressing cells showed increased uptake of DAPI as compared with cells expressing wild-type Cx46, suggesting aberrant connexin hemi-channel activity.Time-lapse and dye uptake studies suggested that T19M hemi-channels had reduced sensitivity to Ca(2+).

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL, 60064, USA.

ABSTRACT
An N-terminal mutant of connexin46 (T19M) alters a highly conserved threonine and has been linked to autosomal dominant cataracts. To study the cellular and functional consequences of substitution of this amino acid, T19M was expressed in Xenopus oocytes and in HeLa cells. Unlike wild-type Cx46, T19M did not induce intercellular conductances in Xenopus oocytes. In transfected HeLa cells, T19M was largely localized within the cytoplasm, with drastically reduced formation of gap junction plaques. Expression of rat T19M was cytotoxic, as evidenced by an almost complete loss of viable cells expressing the mutant protein by 48-72 h following transfection. When incubated in medium containing physiological concentrations of divalent cations, T19M-expressing cells showed increased uptake of DAPI as compared with cells expressing wild-type Cx46, suggesting aberrant connexin hemi-channel activity. Time-lapse and dye uptake studies suggested that T19M hemi-channels had reduced sensitivity to Ca(2+). Whole cell patch clamp studies of single transfected HeLa cells demonstrated that rat T19M formed functional hemi-channels with altered voltage-dependent gating. These data suggest that T19M causes cataracts by loss of gap junctional channel function and abnormally increased hemi-channel activity. Furthermore, they implicate this conserved threonine in both gap junction plaque formation and channel/hemi-channel gating in Cx46.

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T19M is inefficient at forming gap junction plaques. Photomicrographs show the distribution of wild-type rat Cx46 (a) and T19M (b–d) at the indicated times following transient transfection of HeLa cells. Bar 30 μM
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Fig2: T19M is inefficient at forming gap junction plaques. Photomicrographs show the distribution of wild-type rat Cx46 (a) and T19M (b–d) at the indicated times following transient transfection of HeLa cells. Bar 30 μM

Mentions: To determine the ability of T19M to form gap junction plaques, we performed immunofluorescence staining on transiently transfected HeLa cells. Cells transfected with wild-type rat Cx46 showed strong staining at gap junction plaques and some staining in the perinuclear region, likely corresponding to the Golgi apparatus (Fig. 2a). Surprisingly, in multiple independent experiments we detected very few cells containing immunoreactive Cx46 after transfection with rat T19M at our usual time of observation (48 h). Eighteen hours following transfection, most Cx46T19M-expressing cells showed a normal morphology with strong perinuclear staining and no gap junctional plaques (Fig. 2b). By 24 h after transfection, cells had started to round up and the abundance of cells expressing the Cx46 mutant was decreased (Fig. 2c). After 48–72 h, cells showed low intensity of Cx46T19M immunoreactivity, lacked detectable gap junctions, and were rounded up (Fig. 2d). The striking decline in numbers of T19M-expressing cells and their rounded morphology suggested that the mutated protein had toxic effects.Fig. 2


The connexin46 mutant, Cx46T19M, causes loss of gap junction function and alters hemi-channel gating.

Tong JJ, Minogue PJ, Kobeszko M, Beyer EC, Berthoud VM, Ebihara L - J. Membr. Biol. (2014)

T19M is inefficient at forming gap junction plaques. Photomicrographs show the distribution of wild-type rat Cx46 (a) and T19M (b–d) at the indicated times following transient transfection of HeLa cells. Bar 30 μM
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4300453&req=5

Fig2: T19M is inefficient at forming gap junction plaques. Photomicrographs show the distribution of wild-type rat Cx46 (a) and T19M (b–d) at the indicated times following transient transfection of HeLa cells. Bar 30 μM
Mentions: To determine the ability of T19M to form gap junction plaques, we performed immunofluorescence staining on transiently transfected HeLa cells. Cells transfected with wild-type rat Cx46 showed strong staining at gap junction plaques and some staining in the perinuclear region, likely corresponding to the Golgi apparatus (Fig. 2a). Surprisingly, in multiple independent experiments we detected very few cells containing immunoreactive Cx46 after transfection with rat T19M at our usual time of observation (48 h). Eighteen hours following transfection, most Cx46T19M-expressing cells showed a normal morphology with strong perinuclear staining and no gap junctional plaques (Fig. 2b). By 24 h after transfection, cells had started to round up and the abundance of cells expressing the Cx46 mutant was decreased (Fig. 2c). After 48–72 h, cells showed low intensity of Cx46T19M immunoreactivity, lacked detectable gap junctions, and were rounded up (Fig. 2d). The striking decline in numbers of T19M-expressing cells and their rounded morphology suggested that the mutated protein had toxic effects.Fig. 2

Bottom Line: In transfected HeLa cells, T19M was largely localized within the cytoplasm, with drastically reduced formation of gap junction plaques.When incubated in medium containing physiological concentrations of divalent cations, T19M-expressing cells showed increased uptake of DAPI as compared with cells expressing wild-type Cx46, suggesting aberrant connexin hemi-channel activity.Time-lapse and dye uptake studies suggested that T19M hemi-channels had reduced sensitivity to Ca(2+).

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL, 60064, USA.

ABSTRACT
An N-terminal mutant of connexin46 (T19M) alters a highly conserved threonine and has been linked to autosomal dominant cataracts. To study the cellular and functional consequences of substitution of this amino acid, T19M was expressed in Xenopus oocytes and in HeLa cells. Unlike wild-type Cx46, T19M did not induce intercellular conductances in Xenopus oocytes. In transfected HeLa cells, T19M was largely localized within the cytoplasm, with drastically reduced formation of gap junction plaques. Expression of rat T19M was cytotoxic, as evidenced by an almost complete loss of viable cells expressing the mutant protein by 48-72 h following transfection. When incubated in medium containing physiological concentrations of divalent cations, T19M-expressing cells showed increased uptake of DAPI as compared with cells expressing wild-type Cx46, suggesting aberrant connexin hemi-channel activity. Time-lapse and dye uptake studies suggested that T19M hemi-channels had reduced sensitivity to Ca(2+). Whole cell patch clamp studies of single transfected HeLa cells demonstrated that rat T19M formed functional hemi-channels with altered voltage-dependent gating. These data suggest that T19M causes cataracts by loss of gap junctional channel function and abnormally increased hemi-channel activity. Furthermore, they implicate this conserved threonine in both gap junction plaque formation and channel/hemi-channel gating in Cx46.

Show MeSH
Related in: MedlinePlus