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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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Effect of lanthanum ions. Currents before (a) and after the application of La3+ (b; 200 µM) recorded from a HeLa cell expressing T19M. Families of current traces were recorded in response to a series of voltage clamp steps between −60 and 70 mV in increments of 10 mV from a holding potential of −60 mV. Dashed line indicates zero current level. c I–V relations obtained from the data shown in (a, b). The current was measured at the end of the 1-s pulse and plotted as a function of voltage. The concentrations of divalent cations in the bath solution were reduced to zero added Ca2+ and 0.5 mM Mg2+ to augment the size of the hemi-channel currents. dBar graph summarizes the input conductance measured at −60 mV in HeLa cells expressing wild-type Cx46, T19M, or vector (alone) when exposed to extracellular solutions containing 0 mM Ca2+, 0.5 mM Mg2+ (black bars) or 0 mM Ca2+, 0.5 mM Mg2+, 0.2 mM La3+ (gray bars). The number of cells analyzed is indicated within parentheses
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Fig7: Effect of lanthanum ions. Currents before (a) and after the application of La3+ (b; 200 µM) recorded from a HeLa cell expressing T19M. Families of current traces were recorded in response to a series of voltage clamp steps between −60 and 70 mV in increments of 10 mV from a holding potential of −60 mV. Dashed line indicates zero current level. c I–V relations obtained from the data shown in (a, b). The current was measured at the end of the 1-s pulse and plotted as a function of voltage. The concentrations of divalent cations in the bath solution were reduced to zero added Ca2+ and 0.5 mM Mg2+ to augment the size of the hemi-channel currents. dBar graph summarizes the input conductance measured at −60 mV in HeLa cells expressing wild-type Cx46, T19M, or vector (alone) when exposed to extracellular solutions containing 0 mM Ca2+, 0.5 mM Mg2+ (black bars) or 0 mM Ca2+, 0.5 mM Mg2+, 0.2 mM La3+ (gray bars). The number of cells analyzed is indicated within parentheses

Mentions: To determine if the small, persistent inward current observed at negative potentials in T19M-expressing cells was due to hemi-channels, we used the nonspecific hemi-channel blocker, La+3 (John et al. 1999; Contreras et al. 2002). Application of 200 μM La3+ blocked most of the time- and voltage-dependent component of the current (Fig. 7). It also reduced the inward current at the holding potential and decreased the cell input conductance to values comparable to those observed in vector-transfected control cells. Similar results were obtained for wild-type Cx46. The Cx46 hemi-channel currents could also be partially blocked by 200 μM carbenoxolone. These findings suggest that a small number of Cx46 hemi-channels remain open even in the presence of divalent cations and that these channels can account for the dye uptake observed in the presence of divalent cations.Fig. 7


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)

Effect of lanthanum ions. Currents before (a) and after the application of La3+ (b; 200 µM) recorded from a HeLa cell expressing T19M. Families of current traces were recorded in response to a series of voltage clamp steps between −60 and 70 mV in increments of 10 mV from a holding potential of −60 mV. Dashed line indicates zero current level. c I–V relations obtained from the data shown in (a, b). The current was measured at the end of the 1-s pulse and plotted as a function of voltage. The concentrations of divalent cations in the bath solution were reduced to zero added Ca2+ and 0.5 mM Mg2+ to augment the size of the hemi-channel currents. dBar graph summarizes the input conductance measured at −60 mV in HeLa cells expressing wild-type Cx46, T19M, or vector (alone) when exposed to extracellular solutions containing 0 mM Ca2+, 0.5 mM Mg2+ (black bars) or 0 mM Ca2+, 0.5 mM Mg2+, 0.2 mM La3+ (gray bars). The number of cells analyzed is indicated within parentheses
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4300453&req=5

Fig7: Effect of lanthanum ions. Currents before (a) and after the application of La3+ (b; 200 µM) recorded from a HeLa cell expressing T19M. Families of current traces were recorded in response to a series of voltage clamp steps between −60 and 70 mV in increments of 10 mV from a holding potential of −60 mV. Dashed line indicates zero current level. c I–V relations obtained from the data shown in (a, b). The current was measured at the end of the 1-s pulse and plotted as a function of voltage. The concentrations of divalent cations in the bath solution were reduced to zero added Ca2+ and 0.5 mM Mg2+ to augment the size of the hemi-channel currents. dBar graph summarizes the input conductance measured at −60 mV in HeLa cells expressing wild-type Cx46, T19M, or vector (alone) when exposed to extracellular solutions containing 0 mM Ca2+, 0.5 mM Mg2+ (black bars) or 0 mM Ca2+, 0.5 mM Mg2+, 0.2 mM La3+ (gray bars). The number of cells analyzed is indicated within parentheses
Mentions: To determine if the small, persistent inward current observed at negative potentials in T19M-expressing cells was due to hemi-channels, we used the nonspecific hemi-channel blocker, La+3 (John et al. 1999; Contreras et al. 2002). Application of 200 μM La3+ blocked most of the time- and voltage-dependent component of the current (Fig. 7). It also reduced the inward current at the holding potential and decreased the cell input conductance to values comparable to those observed in vector-transfected control cells. Similar results were obtained for wild-type Cx46. The Cx46 hemi-channel currents could also be partially blocked by 200 μM carbenoxolone. These findings suggest that a small number of Cx46 hemi-channels remain open even in the presence of divalent cations and that these channels can account for the dye uptake observed in the presence of divalent cations.Fig. 7

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