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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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The rate of DAPI uptake is increased by lowering divalent cations and inhibited by La3+. Average time course of DAPI uptake by transfected HeLa cells in control solution (1 mM Ca2+, 1 mM Mg2+), in external solutions with no added divalent cations and in control solution plus 200 µM La3+. a Wild-type rat Cx46 (closed circles); T19M (open triangles). To measure changes in the rate of dye uptake over time, the mean DAPI fluorescence intensity per pixel from ROI’s located in the nuclei of Zaza-green positive cells were normalized to mean DAPI fluorescence intensity of the ROI’s at 60 min, averaged and plotted as a function of time. The cells were initially bathed in control solution (containing 1 mM Ca2+, 1 mM Mg2+). Then, the cells were exposed to a solution containing no added divalent cations followed by reperfusion with control solution containing 200 μM La3+. All the solutions contained 4 μM DAPI. bBar graph shows the rates of DAPI uptake in cells expressing wild-type Cx46, T19M, or vector alone in the presence of 1 mM Ca2+, 1 mM Mg2+ (gray bar); or 1 mM Ca2+, 1 mM Mg2+, 0.2 mM La3+ (black bar). Data are presented as the mean ± SEM. *p < 0.002 (Mann–Whitney rank sum test compared with T19M-transfected cells); +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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Fig4: The rate of DAPI uptake is increased by lowering divalent cations and inhibited by La3+. Average time course of DAPI uptake by transfected HeLa cells in control solution (1 mM Ca2+, 1 mM Mg2+), in external solutions with no added divalent cations and in control solution plus 200 µM La3+. a Wild-type rat Cx46 (closed circles); T19M (open triangles). To measure changes in the rate of dye uptake over time, the mean DAPI fluorescence intensity per pixel from ROI’s located in the nuclei of Zaza-green positive cells were normalized to mean DAPI fluorescence intensity of the ROI’s at 60 min, averaged and plotted as a function of time. The cells were initially bathed in control solution (containing 1 mM Ca2+, 1 mM Mg2+). Then, the cells were exposed to a solution containing no added divalent cations followed by reperfusion with control solution containing 200 μM La3+. All the solutions contained 4 μM DAPI. bBar graph shows the rates of DAPI uptake in cells expressing wild-type Cx46, T19M, or vector alone in the presence of 1 mM Ca2+, 1 mM Mg2+ (gray bar); or 1 mM Ca2+, 1 mM Mg2+, 0.2 mM La3+ (black bar). Data are presented as the mean ± SEM. *p < 0.002 (Mann–Whitney rank sum test compared with T19M-transfected cells); +p < 0.001 (Mann–Whitney rank sum test compared with vector-transfected cells). The number of cells analyzed is indicated within parentheses

Mentions: To examine the effect of wild-type and mutant Cx46 on hemi-channel activity in greater detail, we measured the uptake of DAPI by time-lapse recordings in HeLa cells expressing wild-type rat Cx46, T19M, or vector alone (Fig. 4). In the presence of control external solution (containing 1 mM Ca2+and 1 mM Mg2+), cells expressing T19M showed significantly higher rates of DAPI uptake than those expressing wild-type Cx46 or vector alone. After changing the bathing medium to a solution free of divalent cations, the rate of DAPI uptake increased by approximately twofold in the T19M cells and by up to 19-fold in the Cx46 cells. Little or no increase in dye uptake was observed in control cells expressing vector alone (data not shown). Dye uptake was completely blocked by application of 200 μM lanthanum, a nonspecific connexin hemi-channel blocker. Dye uptake was also ~90 % blocked in Cx46 cells and 77 % blocked in T19M cells by application of 500 μM carbenoxolone. These results suggest that the T19M channels are less sensitive to blockade by divalent cations than wild-type Cx46 hemi-channels. Interestingly, the effect of divalent cation-free conditions on the wild-type Cx46 cells was slow, requiring greater than 15 min to reach a new steady-state. In contrast, the effect of divalent cation-free conditions on the T19M cells was relatively fast, reaching steady-state in <2 min.Fig. 4


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)

The rate of DAPI uptake is increased by lowering divalent cations and inhibited by La3+. Average time course of DAPI uptake by transfected HeLa cells in control solution (1 mM Ca2+, 1 mM Mg2+), in external solutions with no added divalent cations and in control solution plus 200 µM La3+. a Wild-type rat Cx46 (closed circles); T19M (open triangles). To measure changes in the rate of dye uptake over time, the mean DAPI fluorescence intensity per pixel from ROI’s located in the nuclei of Zaza-green positive cells were normalized to mean DAPI fluorescence intensity of the ROI’s at 60 min, averaged and plotted as a function of time. The cells were initially bathed in control solution (containing 1 mM Ca2+, 1 mM Mg2+). Then, the cells were exposed to a solution containing no added divalent cations followed by reperfusion with control solution containing 200 μM La3+. All the solutions contained 4 μM DAPI. bBar graph shows the rates of DAPI uptake in cells expressing wild-type Cx46, T19M, or vector alone in the presence of 1 mM Ca2+, 1 mM Mg2+ (gray bar); or 1 mM Ca2+, 1 mM Mg2+, 0.2 mM La3+ (black bar). Data are presented as the mean ± SEM. *p < 0.002 (Mann–Whitney rank sum test compared with T19M-transfected cells); +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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Related In: Results  -  Collection

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Fig4: The rate of DAPI uptake is increased by lowering divalent cations and inhibited by La3+. Average time course of DAPI uptake by transfected HeLa cells in control solution (1 mM Ca2+, 1 mM Mg2+), in external solutions with no added divalent cations and in control solution plus 200 µM La3+. a Wild-type rat Cx46 (closed circles); T19M (open triangles). To measure changes in the rate of dye uptake over time, the mean DAPI fluorescence intensity per pixel from ROI’s located in the nuclei of Zaza-green positive cells were normalized to mean DAPI fluorescence intensity of the ROI’s at 60 min, averaged and plotted as a function of time. The cells were initially bathed in control solution (containing 1 mM Ca2+, 1 mM Mg2+). Then, the cells were exposed to a solution containing no added divalent cations followed by reperfusion with control solution containing 200 μM La3+. All the solutions contained 4 μM DAPI. bBar graph shows the rates of DAPI uptake in cells expressing wild-type Cx46, T19M, or vector alone in the presence of 1 mM Ca2+, 1 mM Mg2+ (gray bar); or 1 mM Ca2+, 1 mM Mg2+, 0.2 mM La3+ (black bar). Data are presented as the mean ± SEM. *p < 0.002 (Mann–Whitney rank sum test compared with T19M-transfected cells); +p < 0.001 (Mann–Whitney rank sum test compared with vector-transfected cells). The number of cells analyzed is indicated within parentheses
Mentions: To examine the effect of wild-type and mutant Cx46 on hemi-channel activity in greater detail, we measured the uptake of DAPI by time-lapse recordings in HeLa cells expressing wild-type rat Cx46, T19M, or vector alone (Fig. 4). In the presence of control external solution (containing 1 mM Ca2+and 1 mM Mg2+), cells expressing T19M showed significantly higher rates of DAPI uptake than those expressing wild-type Cx46 or vector alone. After changing the bathing medium to a solution free of divalent cations, the rate of DAPI uptake increased by approximately twofold in the T19M cells and by up to 19-fold in the Cx46 cells. Little or no increase in dye uptake was observed in control cells expressing vector alone (data not shown). Dye uptake was completely blocked by application of 200 μM lanthanum, a nonspecific connexin hemi-channel blocker. Dye uptake was also ~90 % blocked in Cx46 cells and 77 % blocked in T19M cells by application of 500 μM carbenoxolone. These results suggest that the T19M channels are less sensitive to blockade by divalent cations than wild-type Cx46 hemi-channels. Interestingly, the effect of divalent cation-free conditions on the wild-type Cx46 cells was slow, requiring greater than 15 min to reach a new steady-state. In contrast, the effect of divalent cation-free conditions on the T19M cells was relatively fast, reaching steady-state in <2 min.Fig. 4

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