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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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Untagged Q49X failed to establish functional gap-junction coupling and dominantly inhibited the coupling of co-expressed Cx40 or Cx43 in N2A cells. (A) N2A cell pairs expressing Q49X-IRES-GFP exhibited no coupling (Gj=0 nS), unlike cell pairs expressing Cx40-IRES-GFP (***P<0.001). (B) Cell pairs co-expressing Q49X-IRES-GFP and Cx40-RFP exhibited significantly lower Gj (***P<0.001) compared with the cell pairs co-expressing Cx40-IRES-GFP and Cx40-RFP. (C) Cell pairs co-expressing Q49X-IRES-GFP and Cx43-RFP exhibited a significantly lower Gj (***P<0.001) than that obtained from the cell pairs co-expressing Cx40-IRES-GFP and Cx43-RFP. Number of cell pairs tested in each case is indicated.
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f7-0070561: Untagged Q49X failed to establish functional gap-junction coupling and dominantly inhibited the coupling of co-expressed Cx40 or Cx43 in N2A cells. (A) N2A cell pairs expressing Q49X-IRES-GFP exhibited no coupling (Gj=0 nS), unlike cell pairs expressing Cx40-IRES-GFP (***P<0.001). (B) Cell pairs co-expressing Q49X-IRES-GFP and Cx40-RFP exhibited significantly lower Gj (***P<0.001) compared with the cell pairs co-expressing Cx40-IRES-GFP and Cx40-RFP. (C) Cell pairs co-expressing Q49X-IRES-GFP and Cx43-RFP exhibited a significantly lower Gj (***P<0.001) than that obtained from the cell pairs co-expressing Cx40-IRES-GFP and Cx43-RFP. Number of cell pairs tested in each case is indicated.

Mentions: To eliminate the possibility of the observed dominant-negative properties of the Q49X mutant was due to its YFP tag, the untagged Q49X mutant was co-expressed with fluorescent-protein-tagged Cx40 or Cx43. As shown in Fig. 7, cell pairs co-expressing the Q49X mutant and, via the bicistronic vector, GFP were completely uncoupled. Consistent with the results from the expression of the Q49X-YFP mutant, untagged Q49X also reduced the Gj when it was co-expressed with Cx40 or Cx43 (Fig. 7B,C). These results further support the finding that the loss-of-function Cx40 mutant was also dominant-negative to the function of both Cx40 and Cx43.


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 failed to establish functional gap-junction coupling and dominantly inhibited the coupling of co-expressed Cx40 or Cx43 in N2A cells. (A) N2A cell pairs expressing Q49X-IRES-GFP exhibited no coupling (Gj=0 nS), unlike cell pairs expressing Cx40-IRES-GFP (***P<0.001). (B) Cell pairs co-expressing Q49X-IRES-GFP and Cx40-RFP exhibited significantly lower Gj (***P<0.001) compared with the cell pairs co-expressing Cx40-IRES-GFP and Cx40-RFP. (C) Cell pairs co-expressing Q49X-IRES-GFP and Cx43-RFP exhibited a significantly lower Gj (***P<0.001) than that obtained from the cell pairs co-expressing Cx40-IRES-GFP and Cx43-RFP. Number of cell pairs tested in each case is indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7-0070561: Untagged Q49X failed to establish functional gap-junction coupling and dominantly inhibited the coupling of co-expressed Cx40 or Cx43 in N2A cells. (A) N2A cell pairs expressing Q49X-IRES-GFP exhibited no coupling (Gj=0 nS), unlike cell pairs expressing Cx40-IRES-GFP (***P<0.001). (B) Cell pairs co-expressing Q49X-IRES-GFP and Cx40-RFP exhibited significantly lower Gj (***P<0.001) compared with the cell pairs co-expressing Cx40-IRES-GFP and Cx40-RFP. (C) Cell pairs co-expressing Q49X-IRES-GFP and Cx43-RFP exhibited a significantly lower Gj (***P<0.001) than that obtained from the cell pairs co-expressing Cx40-IRES-GFP and Cx43-RFP. Number of cell pairs tested in each case is indicated.
Mentions: To eliminate the possibility of the observed dominant-negative properties of the Q49X mutant was due to its YFP tag, the untagged Q49X mutant was co-expressed with fluorescent-protein-tagged Cx40 or Cx43. As shown in Fig. 7, cell pairs co-expressing the Q49X mutant and, via the bicistronic vector, GFP were completely uncoupled. Consistent with the results from the expression of the Q49X-YFP mutant, untagged Q49X also reduced the Gj when it was co-expressed with Cx40 or Cx43 (Fig. 7B,C). These results further support the finding that the loss-of-function Cx40 mutant was also dominant-negative to the function of both Cx40 and Cx43.

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