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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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Representative families of current traces recorded from single HeLa cells transfected with vector alone (a), wild-type rat Cx46 (b), or T19M (c). Families of current traces were recorded in response to a series of voltage clamp steps between −60 and 50 mV in increments of 10 mV from a holding potential of −60 mV. Dashed line indicates zero current level. d Average steady-state I–V relationships for vector alone (open squares, n = 5), wild-type (solid circles, n = 4), and T19M (open triangles, n = 3)
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Fig5: Representative families of current traces recorded from single HeLa cells transfected with vector alone (a), wild-type rat Cx46 (b), or T19M (c). Families of current traces were recorded in response to a series of voltage clamp steps between −60 and 50 mV in increments of 10 mV from a holding potential of −60 mV. Dashed line indicates zero current level. d Average steady-state I–V relationships for vector alone (open squares, n = 5), wild-type (solid circles, n = 4), and T19M (open triangles, n = 3)

Mentions: To determine the effects of T19M on plasma membrane conductance of HeLa cells, whole cell patch clamp experiments were performed with cesium in the pipette and sodium in the bath. Figure 5 compares representative families of current traces (and averaged steady-state I–V relationships) recorded from HeLa cells expressing Cx46, T19M, or Zaza green alone in the presence of 1 mM [Ca+2]o and 1 mM [Mg+2]o. Both Cx46- and T19M-expressing cells exhibited currents that were mostly closed at −60 mV and activated in response to depolarizing voltage clamp steps in a time- and voltage-dependent manner. These currents were not observed in either non-transfected cells or Zaza-green-transfected cells, indicating that they could be attributed to Cx46 and T19M hemi-channels. Both wild-type and mutant currents could be readily observed in cells transfected with similar amounts of DNA even when the bath solution contained 1 mM Ca2+ (and 1 mM Mg2+). However, the T19M mutation altered the kinetics of channel gating. T19M currents activated more rapidly and had a more pronounced and prolonged inactivation phase than the wild-type Cx46 currents at large positive potentials as illustrated in Fig. 6a and b. In addition, the time course of deactivation of the T19M currents at negative potentials was slower than that of wild-type Cx46. This effect was quantified by measuring the time required for the tail current to decay to 50 % of its peak value (t1/2). Over the transmembrane voltage (Vm) range between −80 and −40 mV, the average t1/2 values were significantly longer for T19M than for wild-type Cx46 (Fig. 6d). This effect did not appear to correlate with changes in current density since the amplitudes of the T19M tail currents used in the data analysis were smaller than those of the WT tail currents (Fig. 6c).Fig. 5


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)

Representative families of current traces recorded from single HeLa cells transfected with vector alone (a), wild-type rat Cx46 (b), or T19M (c). Families of current traces were recorded in response to a series of voltage clamp steps between −60 and 50 mV in increments of 10 mV from a holding potential of −60 mV. Dashed line indicates zero current level. d Average steady-state I–V relationships for vector alone (open squares, n = 5), wild-type (solid circles, n = 4), and T19M (open triangles, n = 3)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4300453&req=5

Fig5: Representative families of current traces recorded from single HeLa cells transfected with vector alone (a), wild-type rat Cx46 (b), or T19M (c). Families of current traces were recorded in response to a series of voltage clamp steps between −60 and 50 mV in increments of 10 mV from a holding potential of −60 mV. Dashed line indicates zero current level. d Average steady-state I–V relationships for vector alone (open squares, n = 5), wild-type (solid circles, n = 4), and T19M (open triangles, n = 3)
Mentions: To determine the effects of T19M on plasma membrane conductance of HeLa cells, whole cell patch clamp experiments were performed with cesium in the pipette and sodium in the bath. Figure 5 compares representative families of current traces (and averaged steady-state I–V relationships) recorded from HeLa cells expressing Cx46, T19M, or Zaza green alone in the presence of 1 mM [Ca+2]o and 1 mM [Mg+2]o. Both Cx46- and T19M-expressing cells exhibited currents that were mostly closed at −60 mV and activated in response to depolarizing voltage clamp steps in a time- and voltage-dependent manner. These currents were not observed in either non-transfected cells or Zaza-green-transfected cells, indicating that they could be attributed to Cx46 and T19M hemi-channels. Both wild-type and mutant currents could be readily observed in cells transfected with similar amounts of DNA even when the bath solution contained 1 mM Ca2+ (and 1 mM Mg2+). However, the T19M mutation altered the kinetics of channel gating. T19M currents activated more rapidly and had a more pronounced and prolonged inactivation phase than the wild-type Cx46 currents at large positive potentials as illustrated in Fig. 6a and b. In addition, the time course of deactivation of the T19M currents at negative potentials was slower than that of wild-type Cx46. This effect was quantified by measuring the time required for the tail current to decay to 50 % of its peak value (t1/2). Over the transmembrane voltage (Vm) range between −80 and −40 mV, the average t1/2 values were significantly longer for T19M than for wild-type Cx46 (Fig. 6d). This effect did not appear to correlate with changes in current density since the amplitudes of the T19M tail currents used in the data analysis were smaller than those of the WT tail currents (Fig. 6c).Fig. 5

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