Limits...
An atrial-fibrillation-linked connexin40 mutant is retained in the endoplasmic reticulum and impairs the function of atrial gap-junction channels.

Sun Y, Tong X, Chen H, Huang T, Shao Q, Huang W, Laird DW, Bai D - Dis Model Mech (2014)

Bottom Line: When the Q49X mutant was co-expressed with Cx40 or Cx43, the mutant substantially reduced the gap-junction plaque formation of Cx40 and Cx43.Electrophysiological studies revealed no electrical coupling of cell pairs expressing the mutant alone and a significant decrease in the coupling conductance when the mutant was co-expressed with Cx40 or Cx43.Further colocalization experiments with the organelle residential proteins indicate that Q49X was retained in the endoplasmic reticulum.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, The University of Western Ontario, London, ON N6A 5C1, Canada.

ABSTRACT
Connexin40 (Cx40)-containing gap-junction channels are expressed in the atrial myocardium and provide a low-resistance passage for rapid impulse propagation. A germline mutation in the GJA5 gene, which encodes Cx40, resulting in a truncated Cx40 (Q49X) was identified in a large Chinese family with lone (idiopathic) atrial fibrillation (AF). This mutation co-segregated with seven AF probands in an autosomal-dominant way over generations. To test the hypothesis that this Cx40 mutant affects the distribution and function of atrial gap junctions, we studied the Q49X mutant in gap-junction-deficient HeLa and N2A cells. The Q49X mutant, unlike wild-type Cx40, was typically localized in the cytoplasm and failed to form gap-junction plaques at cell-cell interfaces. When the Q49X mutant was co-expressed with Cx40 or Cx43, the mutant substantially reduced the gap-junction plaque formation of Cx40 and Cx43. Electrophysiological studies revealed no electrical coupling of cell pairs expressing the mutant alone and a significant decrease in the coupling conductance when the mutant was co-expressed with Cx40 or Cx43. Further colocalization experiments with the organelle residential proteins indicate that Q49X was retained in the endoplasmic reticulum. These findings provide evidence that the Q49X mutant is capable of impairing gap-junction distribution and function of key atrial connexins, which might play a role in the predisposition to and onset of AF.

Show MeSH

Related in: MedlinePlus

Untagged Q49X mutant expression reduced the incidence of Cx43 gap-junction plaques in NRK cells. NRK cells expressing the untagged Q49X mutant (green) were immunolabeled for Cx43 (red) before (A,B) and after (C,D) treatment with cycloheximide (30 μg/ml for 9 hours). The incidence of Cx43 gap-junction plaques between Q49X-expressing cells (green) was reduced in both conditions. B and D are from the same cells shown in A and C, respectively, but with Hoechst nuclei staining. Asterisks (A,B) indicate Q49X-expressing cells with an obvious reduction of intracellular Cx43. Scale bar: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4007407&req=5

f8-0070561: Untagged Q49X mutant expression reduced the incidence of Cx43 gap-junction plaques in NRK cells. NRK cells expressing the untagged Q49X mutant (green) were immunolabeled for Cx43 (red) before (A,B) and after (C,D) treatment with cycloheximide (30 μg/ml for 9 hours). The incidence of Cx43 gap-junction plaques between Q49X-expressing cells (green) was reduced in both conditions. B and D are from the same cells shown in A and C, respectively, but with Hoechst nuclei staining. Asterisks (A,B) indicate Q49X-expressing cells with an obvious reduction of intracellular Cx43. Scale bar: 20 μm.

Mentions: To study whether the untagged Cx40 Q49X mutant is also able to alter the localization of endogenously expressed Cx43, we expressed the Q49X mutant in normal rat kidney (NRK) cells. Anti-Cx43 antibody labeling showed an abundant expression of Cx43 in the intracellular compartments and at the cell-cell interfaces in non-transfected NRK cells (Fig. 8A). Expression of the Q49X mutant (green cells in Fig. 8A) substantially reduced the apparent number of Cx43 gap-junction plaques at the interfaces between mutant-expressing cells (Fig. 8). Reduction of intracellular Cx43 was also observed in some cells expressing the mutant, especially in the perinuclear area (cells with asterisks in Fig. 8A,B). As expected, a 9-hour treatment of NRK cells with a protein-synthesis inhibitor, cycloheximide, substantially reduced the overall level of Cx43, especially in the intracellular compartments (Fig. 8C,D), yet Cx43 plaques were still prominent at the cell-cell interfaces. Similar to the effects of the Q49X mutant in the absence of cycloheximide, the mutant reduced the size and/or number of gap-junction plaques at the cell-cell interfaces between mutant-expressing cells in the presence of cycloheximide (Fig. 8C,D).


An atrial-fibrillation-linked connexin40 mutant is retained in the endoplasmic reticulum and impairs the function of atrial gap-junction channels.

Sun Y, Tong X, Chen H, Huang T, Shao Q, Huang W, Laird DW, Bai D - Dis Model Mech (2014)

Untagged Q49X mutant expression reduced the incidence of Cx43 gap-junction plaques in NRK cells. NRK cells expressing the untagged Q49X mutant (green) were immunolabeled for Cx43 (red) before (A,B) and after (C,D) treatment with cycloheximide (30 μg/ml for 9 hours). The incidence of Cx43 gap-junction plaques between Q49X-expressing cells (green) was reduced in both conditions. B and D are from the same cells shown in A and C, respectively, but with Hoechst nuclei staining. Asterisks (A,B) indicate Q49X-expressing cells with an obvious reduction of intracellular Cx43. Scale bar: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4007407&req=5

f8-0070561: Untagged Q49X mutant expression reduced the incidence of Cx43 gap-junction plaques in NRK cells. NRK cells expressing the untagged Q49X mutant (green) were immunolabeled for Cx43 (red) before (A,B) and after (C,D) treatment with cycloheximide (30 μg/ml for 9 hours). The incidence of Cx43 gap-junction plaques between Q49X-expressing cells (green) was reduced in both conditions. B and D are from the same cells shown in A and C, respectively, but with Hoechst nuclei staining. Asterisks (A,B) indicate Q49X-expressing cells with an obvious reduction of intracellular Cx43. Scale bar: 20 μm.
Mentions: To study whether the untagged Cx40 Q49X mutant is also able to alter the localization of endogenously expressed Cx43, we expressed the Q49X mutant in normal rat kidney (NRK) cells. Anti-Cx43 antibody labeling showed an abundant expression of Cx43 in the intracellular compartments and at the cell-cell interfaces in non-transfected NRK cells (Fig. 8A). Expression of the Q49X mutant (green cells in Fig. 8A) substantially reduced the apparent number of Cx43 gap-junction plaques at the interfaces between mutant-expressing cells (Fig. 8). Reduction of intracellular Cx43 was also observed in some cells expressing the mutant, especially in the perinuclear area (cells with asterisks in Fig. 8A,B). As expected, a 9-hour treatment of NRK cells with a protein-synthesis inhibitor, cycloheximide, substantially reduced the overall level of Cx43, especially in the intracellular compartments (Fig. 8C,D), yet Cx43 plaques were still prominent at the cell-cell interfaces. Similar to the effects of the Q49X mutant in the absence of cycloheximide, the mutant reduced the size and/or number of gap-junction plaques at the cell-cell interfaces between mutant-expressing cells in the presence of cycloheximide (Fig. 8C,D).

Bottom Line: When the Q49X mutant was co-expressed with Cx40 or Cx43, the mutant substantially reduced the gap-junction plaque formation of Cx40 and Cx43.Electrophysiological studies revealed no electrical coupling of cell pairs expressing the mutant alone and a significant decrease in the coupling conductance when the mutant was co-expressed with Cx40 or Cx43.Further colocalization experiments with the organelle residential proteins indicate that Q49X was retained in the endoplasmic reticulum.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, The University of Western Ontario, London, ON N6A 5C1, Canada.

ABSTRACT
Connexin40 (Cx40)-containing gap-junction channels are expressed in the atrial myocardium and provide a low-resistance passage for rapid impulse propagation. A germline mutation in the GJA5 gene, which encodes Cx40, resulting in a truncated Cx40 (Q49X) was identified in a large Chinese family with lone (idiopathic) atrial fibrillation (AF). This mutation co-segregated with seven AF probands in an autosomal-dominant way over generations. To test the hypothesis that this Cx40 mutant affects the distribution and function of atrial gap junctions, we studied the Q49X mutant in gap-junction-deficient HeLa and N2A cells. The Q49X mutant, unlike wild-type Cx40, was typically localized in the cytoplasm and failed to form gap-junction plaques at cell-cell interfaces. When the Q49X mutant was co-expressed with Cx40 or Cx43, the mutant substantially reduced the gap-junction plaque formation of Cx40 and Cx43. Electrophysiological studies revealed no electrical coupling of cell pairs expressing the mutant alone and a significant decrease in the coupling conductance when the mutant was co-expressed with Cx40 or Cx43. Further colocalization experiments with the organelle residential proteins indicate that Q49X was retained in the endoplasmic reticulum. These findings provide evidence that the Q49X mutant is capable of impairing gap-junction distribution and function of key atrial connexins, which might play a role in the predisposition to and onset of AF.

Show MeSH
Related in: MedlinePlus