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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.

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Subcellular localization of the Q49X mutant in HeLa cells. HeLa cells were engineered to express Cx40-YFP or Q49X-YFP prior to immunolabeling for a resident protein of the Golgi apparatus (GM130) (A) or the ER (PDI) (B). Note that the mutant primarily colocalized with PDI. Scale bars: 20 μm.
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f5-0070561: Subcellular localization of the Q49X mutant in HeLa cells. HeLa cells were engineered to express Cx40-YFP or Q49X-YFP prior to immunolabeling for a resident protein of the Golgi apparatus (GM130) (A) or the ER (PDI) (B). Note that the mutant primarily colocalized with PDI. Scale bars: 20 μm.

Mentions: To determine the subcellular localizations of the mutant protein, Q49X-YFP-expressing HeLa cells were further immunolabeled with antibodies against either PDI, a resident protein of the ER, or GM130, a resident protein of the Golgi apparatus (Fig. 5). Q49X seemed to preferentially colocalize with PDI (Fig. 5B), indicating that the mutant acquires a steady-state residence in the ER. Partial colocalization of wild-type Cx40 (or Q49X) and GM130 was also observed (Fig. 5A). To further confirm the localization of the Q49X mutant to the ER, HeLa cells were engineered to co-express Q49X-YFP and DsRed-ER (live fluorescent marker for ER). Q49X shared a remarkably similar localization pattern with that of DsRed-ER (Pearson’s correlation=0.73±0.06, n=9, Fig. 6C). In contrast, Cx40 exhibited a much weaker colocalization with the ER (Pearson’s correlation=0.18±0.03, n=7, Fig. 6B). Unlike free YFP, the Q49X mutant was not localized to the nucleus (Fig. 6A,C). These findings suggest that the truncated Cx40 mutant Q49X was retained primarily within the ER.


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)

Subcellular localization of the Q49X mutant in HeLa cells. HeLa cells were engineered to express Cx40-YFP or Q49X-YFP prior to immunolabeling for a resident protein of the Golgi apparatus (GM130) (A) or the ER (PDI) (B). Note that the mutant primarily colocalized with PDI. Scale bars: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5-0070561: Subcellular localization of the Q49X mutant in HeLa cells. HeLa cells were engineered to express Cx40-YFP or Q49X-YFP prior to immunolabeling for a resident protein of the Golgi apparatus (GM130) (A) or the ER (PDI) (B). Note that the mutant primarily colocalized with PDI. Scale bars: 20 μm.
Mentions: To determine the subcellular localizations of the mutant protein, Q49X-YFP-expressing HeLa cells were further immunolabeled with antibodies against either PDI, a resident protein of the ER, or GM130, a resident protein of the Golgi apparatus (Fig. 5). Q49X seemed to preferentially colocalize with PDI (Fig. 5B), indicating that the mutant acquires a steady-state residence in the ER. Partial colocalization of wild-type Cx40 (or Q49X) and GM130 was also observed (Fig. 5A). To further confirm the localization of the Q49X mutant to the ER, HeLa cells were engineered to co-express Q49X-YFP and DsRed-ER (live fluorescent marker for ER). Q49X shared a remarkably similar localization pattern with that of DsRed-ER (Pearson’s correlation=0.73±0.06, n=9, Fig. 6C). In contrast, Cx40 exhibited a much weaker colocalization with the ER (Pearson’s correlation=0.18±0.03, n=7, Fig. 6B). Unlike free YFP, the Q49X mutant was not localized to the nucleus (Fig. 6A,C). These findings suggest that the truncated Cx40 mutant Q49X was retained primarily within the ER.

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