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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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Effects of T19M mutation on human Cx46. Photomicrographs showing the distribution of human wild-type Cx46 (a) and T19M (b) in transfected HeLa cells. Bar 30 µm. cBar graph summarizes the DAPI uptake data for vector alone, human wild-type Cx46 and T19M obtained in transfected HeLa cells when exposed to extracellular solutions containing 1 mM Ca2+, 1 mM Mg2+ (gray bars) or 1 mM Ca2+, 1 mM Mg2+, 0.2 mM La3+ (black bars). Data are graphed as mean ± SEM. *p < 0.004 (Mann–Whitney rank sum test compared with T19M-transfected cells in the same external solution); +p < 0.001 (Mann–Whitney rank sum test compared with vector-transfected cells). The number of cells analyzed is indicated within parentheses
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Fig8: Effects of T19M mutation on human Cx46. Photomicrographs showing the distribution of human wild-type Cx46 (a) and T19M (b) in transfected HeLa cells. Bar 30 µm. cBar graph summarizes the DAPI uptake data for vector alone, human wild-type Cx46 and T19M obtained in transfected HeLa cells when exposed to extracellular solutions containing 1 mM Ca2+, 1 mM Mg2+ (gray bars) or 1 mM Ca2+, 1 mM Mg2+, 0.2 mM La3+ (black bars). Data are graphed as mean ± SEM. *p < 0.004 (Mann–Whitney rank sum test compared with T19M-transfected cells in the same external solution); +p < 0.001 (Mann–Whitney rank sum test compared with vector-transfected cells). The number of cells analyzed is indicated within parentheses

Mentions: Because the T19M was originally identified in a human family (Santhiya et al. 2010), we investigated whether human wild-type Cx46 and T19M behaved similarly. Indeed, wild-type human Cx46 localized extensively to gap junction plaques with some localization in the cytoplasm in transfected HeLa cells (Fig. 8a). In contrast, human T19M immunoreactivity was detected mainly in intracellular compartments; on very rare occasions a faint staining at appositional membranes was detected (Fig. 8b).Fig. 8


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)

Effects of T19M mutation on human Cx46. Photomicrographs showing the distribution of human wild-type Cx46 (a) and T19M (b) in transfected HeLa cells. Bar 30 µm. cBar graph summarizes the DAPI uptake data for vector alone, human wild-type Cx46 and T19M obtained in transfected HeLa cells when exposed to extracellular solutions containing 1 mM Ca2+, 1 mM Mg2+ (gray bars) or 1 mM Ca2+, 1 mM Mg2+, 0.2 mM La3+ (black bars). Data are graphed as mean ± SEM. *p < 0.004 (Mann–Whitney rank sum test compared with T19M-transfected cells in the same external solution); +p < 0.001 (Mann–Whitney rank sum test compared with vector-transfected cells). The number of cells analyzed is indicated within parentheses
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig8: Effects of T19M mutation on human Cx46. Photomicrographs showing the distribution of human wild-type Cx46 (a) and T19M (b) in transfected HeLa cells. Bar 30 µm. cBar graph summarizes the DAPI uptake data for vector alone, human wild-type Cx46 and T19M obtained in transfected HeLa cells when exposed to extracellular solutions containing 1 mM Ca2+, 1 mM Mg2+ (gray bars) or 1 mM Ca2+, 1 mM Mg2+, 0.2 mM La3+ (black bars). Data are graphed as mean ± SEM. *p < 0.004 (Mann–Whitney rank sum test compared with T19M-transfected cells in the same external solution); +p < 0.001 (Mann–Whitney rank sum test compared with vector-transfected cells). The number of cells analyzed is indicated within parentheses
Mentions: Because the T19M was originally identified in a human family (Santhiya et al. 2010), we investigated whether human wild-type Cx46 and T19M behaved similarly. Indeed, wild-type human Cx46 localized extensively to gap junction plaques with some localization in the cytoplasm in transfected HeLa cells (Fig. 8a). In contrast, human T19M immunoreactivity was detected mainly in intracellular compartments; on very rare occasions a faint staining at appositional membranes was detected (Fig. 8b).Fig. 8

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