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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 causes increased uptake of connexon-permeant dyes. Photomicrographs show examples of HeLa cells that were transfected with wild-type rat Cx46 (a–c) or T19M (d–i) (using the vector PBI-CMV3 which also drives expression of Zaza green) and incubated a day later with DAPI in Na gluconate Ringer’s solution containing 0 mM Ca2+ (a–f) or 5 mM Ca+2 (g–i) for 20 min. Phase contrast images (a, d, g). Zaza-green fluorescence (b, e, h). DAPI fluorescence (c, f, i). After a 20-min incubation in control solution containing 0 mM Ca2+, cells expressing T19M showed DAPI uptake (e, f) that was mostly inhibited by 5 mM Ca2+ (h, i). Bar graph summarizes the quantification of the DAPI uptake data (j). Data are graphed as mean ± SEM. The number of cells tested is indicated within parentheses. *p < 0.001 (Mann–Whitney rank sum test compared with wild-type rat Cx46-transfected cells)
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Fig3: T19M causes increased uptake of connexon-permeant dyes. Photomicrographs show examples of HeLa cells that were transfected with wild-type rat Cx46 (a–c) or T19M (d–i) (using the vector PBI-CMV3 which also drives expression of Zaza green) and incubated a day later with DAPI in Na gluconate Ringer’s solution containing 0 mM Ca2+ (a–f) or 5 mM Ca+2 (g–i) for 20 min. Phase contrast images (a, d, g). Zaza-green fluorescence (b, e, h). DAPI fluorescence (c, f, i). After a 20-min incubation in control solution containing 0 mM Ca2+, cells expressing T19M showed DAPI uptake (e, f) that was mostly inhibited by 5 mM Ca2+ (h, i). Bar graph summarizes the quantification of the DAPI uptake data (j). Data are graphed as mean ± SEM. The number of cells tested is indicated within parentheses. *p < 0.001 (Mann–Whitney rank sum test compared with wild-type rat Cx46-transfected cells)

Mentions: To test the hypothesis that the apparent cell toxicity in T19M-expressing cells was a consequence of aberrant T19M hemi-channel activity, we examined uptake of the connexon-permeant dye, DAPI, in transfected HeLa cells. Cells that expressed rat Cx46 or T19M were identified by the fluorescence of the reporter protein, Zaza green (Fig. 3). When incubated in the absence of divalent cations or in the presence of 1 mM external Ca2+ concentration ([Ca2+]o and 1 mM [Mg2+]o), cells expressing T19M showed significantly increased DAPI uptake compared to cells expressing wild-type Cx46 or control (Zaza-green negative) cells. The T19M-induced dye uptake was mostly blocked by increasing the [Ca+2]o to 5 mM.Fig. 3


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 causes increased uptake of connexon-permeant dyes. Photomicrographs show examples of HeLa cells that were transfected with wild-type rat Cx46 (a–c) or T19M (d–i) (using the vector PBI-CMV3 which also drives expression of Zaza green) and incubated a day later with DAPI in Na gluconate Ringer’s solution containing 0 mM Ca2+ (a–f) or 5 mM Ca+2 (g–i) for 20 min. Phase contrast images (a, d, g). Zaza-green fluorescence (b, e, h). DAPI fluorescence (c, f, i). After a 20-min incubation in control solution containing 0 mM Ca2+, cells expressing T19M showed DAPI uptake (e, f) that was mostly inhibited by 5 mM Ca2+ (h, i). Bar graph summarizes the quantification of the DAPI uptake data (j). Data are graphed as mean ± SEM. The number of cells tested is indicated within parentheses. *p < 0.001 (Mann–Whitney rank sum test compared with wild-type rat Cx46-transfected cells)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4300453&req=5

Fig3: T19M causes increased uptake of connexon-permeant dyes. Photomicrographs show examples of HeLa cells that were transfected with wild-type rat Cx46 (a–c) or T19M (d–i) (using the vector PBI-CMV3 which also drives expression of Zaza green) and incubated a day later with DAPI in Na gluconate Ringer’s solution containing 0 mM Ca2+ (a–f) or 5 mM Ca+2 (g–i) for 20 min. Phase contrast images (a, d, g). Zaza-green fluorescence (b, e, h). DAPI fluorescence (c, f, i). After a 20-min incubation in control solution containing 0 mM Ca2+, cells expressing T19M showed DAPI uptake (e, f) that was mostly inhibited by 5 mM Ca2+ (h, i). Bar graph summarizes the quantification of the DAPI uptake data (j). Data are graphed as mean ± SEM. The number of cells tested is indicated within parentheses. *p < 0.001 (Mann–Whitney rank sum test compared with wild-type rat Cx46-transfected cells)
Mentions: To test the hypothesis that the apparent cell toxicity in T19M-expressing cells was a consequence of aberrant T19M hemi-channel activity, we examined uptake of the connexon-permeant dye, DAPI, in transfected HeLa cells. Cells that expressed rat Cx46 or T19M were identified by the fluorescence of the reporter protein, Zaza green (Fig. 3). When incubated in the absence of divalent cations or in the presence of 1 mM external Ca2+ concentration ([Ca2+]o and 1 mM [Mg2+]o), cells expressing T19M showed significantly increased DAPI uptake compared to cells expressing wild-type Cx46 or control (Zaza-green negative) cells. The T19M-induced dye uptake was mostly blocked by increasing the [Ca+2]o to 5 mM.Fig. 3

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