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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 does not induce gap junctional coupling when expressed by itself, and it acts as a loss-of-function mutation without dominant-negative inhibition when co-expressed with wild-type lens connexins. Bar graphs show mean gap junctional conductances in pairs of oocytes expressing different combinations of wild-type and mutant lens connexins as determined using the double two-electrode voltage clamp technique. a Rat Cx46 or T19M were expressed alone or in combination with each other. b Mouse Cx50 was expressed alone or in combination with either rat Cx46 or T19M. AS indicates oocytes that were injected with no cRNA (i.e., Xenopus Cx38 antisense oligonucleotide alone). The number of pairs tested is indicated within parentheses. *p < 0.001 (Student’s t test compared with Cx46-injected oocyte pairs); **p < 0.001 (Student’s t test compared with Cx50-injected oocyte pairs)
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Fig1: T19M does not induce gap junctional coupling when expressed by itself, and it acts as a loss-of-function mutation without dominant-negative inhibition when co-expressed with wild-type lens connexins. Bar graphs show mean gap junctional conductances in pairs of oocytes expressing different combinations of wild-type and mutant lens connexins as determined using the double two-electrode voltage clamp technique. a Rat Cx46 or T19M were expressed alone or in combination with each other. b Mouse Cx50 was expressed alone or in combination with either rat Cx46 or T19M. AS indicates oocytes that were injected with no cRNA (i.e., Xenopus Cx38 antisense oligonucleotide alone). The number of pairs tested is indicated within parentheses. *p < 0.001 (Student’s t test compared with Cx46-injected oocyte pairs); **p < 0.001 (Student’s t test compared with Cx50-injected oocyte pairs)

Mentions: We initially tested whether T19M was able to induce gap junctional (intercellular) conductances in paired Xenopus oocytes using the double two-electrode voltage clamp technique. Because rodent Cx46 induces higher levels of gap junctional currents than human Cx46 in this system, experiments were initially performed after injecting cRNAs encoding either wild-type or mutant rat Cx46. Homotypic oocyte pairs expressing wild-type rat Cx46 were well coupled, but oocyte pairs injected with rat T19M cRNA showed no coupling above control levels (Fig. 1a). Furthermore, oocytes expressing rat T19M failed to induce significant coupling when paired heterotypically with oocytes expressing wild-type Cx46 (Fig. 1a).Fig. 1


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 does not induce gap junctional coupling when expressed by itself, and it acts as a loss-of-function mutation without dominant-negative inhibition when co-expressed with wild-type lens connexins. Bar graphs show mean gap junctional conductances in pairs of oocytes expressing different combinations of wild-type and mutant lens connexins as determined using the double two-electrode voltage clamp technique. a Rat Cx46 or T19M were expressed alone or in combination with each other. b Mouse Cx50 was expressed alone or in combination with either rat Cx46 or T19M. AS indicates oocytes that were injected with no cRNA (i.e., Xenopus Cx38 antisense oligonucleotide alone). The number of pairs tested is indicated within parentheses. *p < 0.001 (Student’s t test compared with Cx46-injected oocyte pairs); **p < 0.001 (Student’s t test compared with Cx50-injected oocyte pairs)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4300453&req=5

Fig1: T19M does not induce gap junctional coupling when expressed by itself, and it acts as a loss-of-function mutation without dominant-negative inhibition when co-expressed with wild-type lens connexins. Bar graphs show mean gap junctional conductances in pairs of oocytes expressing different combinations of wild-type and mutant lens connexins as determined using the double two-electrode voltage clamp technique. a Rat Cx46 or T19M were expressed alone or in combination with each other. b Mouse Cx50 was expressed alone or in combination with either rat Cx46 or T19M. AS indicates oocytes that were injected with no cRNA (i.e., Xenopus Cx38 antisense oligonucleotide alone). The number of pairs tested is indicated within parentheses. *p < 0.001 (Student’s t test compared with Cx46-injected oocyte pairs); **p < 0.001 (Student’s t test compared with Cx50-injected oocyte pairs)
Mentions: We initially tested whether T19M was able to induce gap junctional (intercellular) conductances in paired Xenopus oocytes using the double two-electrode voltage clamp technique. Because rodent Cx46 induces higher levels of gap junctional currents than human Cx46 in this system, experiments were initially performed after injecting cRNAs encoding either wild-type or mutant rat Cx46. Homotypic oocyte pairs expressing wild-type rat Cx46 were well coupled, but oocyte pairs injected with rat T19M cRNA showed no coupling above control levels (Fig. 1a). Furthermore, oocytes expressing rat T19M failed to induce significant coupling when paired heterotypically with oocytes expressing wild-type Cx46 (Fig. 1a).Fig. 1

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