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Cx43 phosphorylation on S279/282 and intercellular communication are regulated by IP3/IP3 receptor signaling.

Kang M, Lin N, Li C, Meng Q, Zheng Y, Yan X, Deng J, Ou Y, Zhang C, He J, Luo D - Cell Commun. Signal (2014)

Bottom Line: Site-directed mutagenesis indicated that expression of a mutant Cx43-S282A (alanine) inhibited S279/282 phosphorylation and GJ permeability, while the S279A mutant showed the opposite effect in ventricular myocytes.Expression of these mutants in HEK293 cells revealed that cells with a dual S279/282 mutation failed to express exogenous Cx43, whereas cells with a single S279 or S282 mutation displayed Cx43 overexpression with increased phosphorylation of S279/282 and promotion of intercellular communication.These results demonstrated, for the first time, that IP3R physically interacts with Cx43 and participates in the regulation of Cx43 phosphorylation on S279/282, thereby affecting GJ intercellular communication in ventricular myocytes.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Inositol 1,4,5-trisphosphate receptor (IP3R) plays a pivotal role in the Ca2+ release process in a variety of cell types. Additionally, IP3R is distributed in ventricular intercalated discs, but its function(s) in this particular site remains unknown. Connexin (Cx43), the predominant gap junction (GJ) protein in ventricular myocardium, is linked to several signaling pathways that regulate Cx43 properties by (de)phosphorylation on multiple residues. Here, we investigated the regulatory role of IP3R in cell-cell communication and the mechanism(s) underlying this effect.

Results: In neonatal rat and adult mouse ventricular myocytes IP3R co-localized and co-immunoprecipitated with Cx43 in GJ plaques detected by immunostaining and western blot assays. Blocking IP3R with antagonists or silencing pan-IP3R expression with shRNA hindered the 6-carboxyfluorescein (6-CFDA) diffusion through GJs and desynchronized Ca2+ transients among confluent neonatal myocytes in culture, whereas stimulation of IP3R with IP3 ester or ATP exerted the opposite effect. Likewise, 6-CFDA propagation through GJs was modulated by IP3R activation or inhibition in cell pairs of isolated adult cardiomyocytes. Furthermore, IP3R activation or IP3R suppression promoted or suppressed, respectively, Cx43 phosphorylation on S279/282. Site-directed mutagenesis indicated that expression of a mutant Cx43-S282A (alanine) inhibited S279/282 phosphorylation and GJ permeability, while the S279A mutant showed the opposite effect in ventricular myocytes. Expression of these mutants in HEK293 cells revealed that cells with a dual S279/282 mutation failed to express exogenous Cx43, whereas cells with a single S279 or S282 mutation displayed Cx43 overexpression with increased phosphorylation of S279/282 and promotion of intercellular communication.

Conclusions: These results demonstrated, for the first time, that IP3R physically interacts with Cx43 and participates in the regulation of Cx43 phosphorylation on S279/282, thereby affecting GJ intercellular communication in ventricular myocytes.

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Effects of S279 or S282 mutation on Cx43 expression and gap junction permeability in non-muscle cells. Representative western blots of lysates from HEK293 cells (A), which had been transduced with plasmids carrying rat nonspecific sequence (vector), wt-Cx43, S279A, S282A or S279A/282A genes for 48 hours, show the changes in the relative levels of pS279/282 and Cx43 expressions due to the mutations. Here, one blot membrane with 6 left bands and another membrane with 4 right bands from the same lysates were connected together to show all treatment groups. Some groups of the HEK293 cells were also treated with 5 μM 2-APB for 10 minutes, while others were stained with anti-Cx43 antibody to examine the subcellular distribution of Cx43 upon different treatments as indicated (B). Scale bar: 10 μm. The statistical data normalized by β-actin depict the effects of different Cx43 mutants and 2-APB on S279/282 phosphorylation and Cx43 expression (C). N = 4 independent determinations for western blotting and immunostaining tests, respectively. In addition, the statistical data of LY uptake depict the effects of the different mutants on GJ permeability (D). N = 5–6 independent determinations for each bar. *P <0.05 and **P <0.01 vs. DMSO (0.1%); #P <0.05 and ##P <0.01 vs. vector, respectively.
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Fig7: Effects of S279 or S282 mutation on Cx43 expression and gap junction permeability in non-muscle cells. Representative western blots of lysates from HEK293 cells (A), which had been transduced with plasmids carrying rat nonspecific sequence (vector), wt-Cx43, S279A, S282A or S279A/282A genes for 48 hours, show the changes in the relative levels of pS279/282 and Cx43 expressions due to the mutations. Here, one blot membrane with 6 left bands and another membrane with 4 right bands from the same lysates were connected together to show all treatment groups. Some groups of the HEK293 cells were also treated with 5 μM 2-APB for 10 minutes, while others were stained with anti-Cx43 antibody to examine the subcellular distribution of Cx43 upon different treatments as indicated (B). Scale bar: 10 μm. The statistical data normalized by β-actin depict the effects of different Cx43 mutants and 2-APB on S279/282 phosphorylation and Cx43 expression (C). N = 4 independent determinations for western blotting and immunostaining tests, respectively. In addition, the statistical data of LY uptake depict the effects of the different mutants on GJ permeability (D). N = 5–6 independent determinations for each bar. *P <0.05 and **P <0.01 vs. DMSO (0.1%); #P <0.05 and ##P <0.01 vs. vector, respectively.

Mentions: To further determine the role of S279/282 phosphorylation in the regulation of GJ permeability, HEK293 cells (they also possess 2-APB-sensitive endogenous Cx43 [39]) expressing the abovementioned mutants were used to determine the differences in exogenous Cx43 phosphorylation on S279/282 and in GJ permeability. Interestingly, only cells transduced with the S279A/282A mutant failed to increase the Cx43 expression, while all the other cells treated with wt-Cx43, S279A, or S282A mutant displayed elevated S279/282 phosphorylation and Cx43 expression, which was distributed in the cytosol and on the cell surface (Figure 7A-C). Consistently, functional evaluation of GJ permeability showed a linkage of LY uptake with the increased Cx43 phosphorylation on S279/282 and exogenous Cx43 expression, and that 5 μM 2-APB abrogated the S279/282 phosphorylation as well as the LY uptake in the control and all the Cx43-manipulated HEK293 cells (Figure 7D).Figure 7


Cx43 phosphorylation on S279/282 and intercellular communication are regulated by IP3/IP3 receptor signaling.

Kang M, Lin N, Li C, Meng Q, Zheng Y, Yan X, Deng J, Ou Y, Zhang C, He J, Luo D - Cell Commun. Signal (2014)

Effects of S279 or S282 mutation on Cx43 expression and gap junction permeability in non-muscle cells. Representative western blots of lysates from HEK293 cells (A), which had been transduced with plasmids carrying rat nonspecific sequence (vector), wt-Cx43, S279A, S282A or S279A/282A genes for 48 hours, show the changes in the relative levels of pS279/282 and Cx43 expressions due to the mutations. Here, one blot membrane with 6 left bands and another membrane with 4 right bands from the same lysates were connected together to show all treatment groups. Some groups of the HEK293 cells were also treated with 5 μM 2-APB for 10 minutes, while others were stained with anti-Cx43 antibody to examine the subcellular distribution of Cx43 upon different treatments as indicated (B). Scale bar: 10 μm. The statistical data normalized by β-actin depict the effects of different Cx43 mutants and 2-APB on S279/282 phosphorylation and Cx43 expression (C). N = 4 independent determinations for western blotting and immunostaining tests, respectively. In addition, the statistical data of LY uptake depict the effects of the different mutants on GJ permeability (D). N = 5–6 independent determinations for each bar. *P <0.05 and **P <0.01 vs. DMSO (0.1%); #P <0.05 and ##P <0.01 vs. vector, respectively.
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Fig7: Effects of S279 or S282 mutation on Cx43 expression and gap junction permeability in non-muscle cells. Representative western blots of lysates from HEK293 cells (A), which had been transduced with plasmids carrying rat nonspecific sequence (vector), wt-Cx43, S279A, S282A or S279A/282A genes for 48 hours, show the changes in the relative levels of pS279/282 and Cx43 expressions due to the mutations. Here, one blot membrane with 6 left bands and another membrane with 4 right bands from the same lysates were connected together to show all treatment groups. Some groups of the HEK293 cells were also treated with 5 μM 2-APB for 10 minutes, while others were stained with anti-Cx43 antibody to examine the subcellular distribution of Cx43 upon different treatments as indicated (B). Scale bar: 10 μm. The statistical data normalized by β-actin depict the effects of different Cx43 mutants and 2-APB on S279/282 phosphorylation and Cx43 expression (C). N = 4 independent determinations for western blotting and immunostaining tests, respectively. In addition, the statistical data of LY uptake depict the effects of the different mutants on GJ permeability (D). N = 5–6 independent determinations for each bar. *P <0.05 and **P <0.01 vs. DMSO (0.1%); #P <0.05 and ##P <0.01 vs. vector, respectively.
Mentions: To further determine the role of S279/282 phosphorylation in the regulation of GJ permeability, HEK293 cells (they also possess 2-APB-sensitive endogenous Cx43 [39]) expressing the abovementioned mutants were used to determine the differences in exogenous Cx43 phosphorylation on S279/282 and in GJ permeability. Interestingly, only cells transduced with the S279A/282A mutant failed to increase the Cx43 expression, while all the other cells treated with wt-Cx43, S279A, or S282A mutant displayed elevated S279/282 phosphorylation and Cx43 expression, which was distributed in the cytosol and on the cell surface (Figure 7A-C). Consistently, functional evaluation of GJ permeability showed a linkage of LY uptake with the increased Cx43 phosphorylation on S279/282 and exogenous Cx43 expression, and that 5 μM 2-APB abrogated the S279/282 phosphorylation as well as the LY uptake in the control and all the Cx43-manipulated HEK293 cells (Figure 7D).Figure 7

Bottom Line: Site-directed mutagenesis indicated that expression of a mutant Cx43-S282A (alanine) inhibited S279/282 phosphorylation and GJ permeability, while the S279A mutant showed the opposite effect in ventricular myocytes.Expression of these mutants in HEK293 cells revealed that cells with a dual S279/282 mutation failed to express exogenous Cx43, whereas cells with a single S279 or S282 mutation displayed Cx43 overexpression with increased phosphorylation of S279/282 and promotion of intercellular communication.These results demonstrated, for the first time, that IP3R physically interacts with Cx43 and participates in the regulation of Cx43 phosphorylation on S279/282, thereby affecting GJ intercellular communication in ventricular myocytes.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Inositol 1,4,5-trisphosphate receptor (IP3R) plays a pivotal role in the Ca2+ release process in a variety of cell types. Additionally, IP3R is distributed in ventricular intercalated discs, but its function(s) in this particular site remains unknown. Connexin (Cx43), the predominant gap junction (GJ) protein in ventricular myocardium, is linked to several signaling pathways that regulate Cx43 properties by (de)phosphorylation on multiple residues. Here, we investigated the regulatory role of IP3R in cell-cell communication and the mechanism(s) underlying this effect.

Results: In neonatal rat and adult mouse ventricular myocytes IP3R co-localized and co-immunoprecipitated with Cx43 in GJ plaques detected by immunostaining and western blot assays. Blocking IP3R with antagonists or silencing pan-IP3R expression with shRNA hindered the 6-carboxyfluorescein (6-CFDA) diffusion through GJs and desynchronized Ca2+ transients among confluent neonatal myocytes in culture, whereas stimulation of IP3R with IP3 ester or ATP exerted the opposite effect. Likewise, 6-CFDA propagation through GJs was modulated by IP3R activation or inhibition in cell pairs of isolated adult cardiomyocytes. Furthermore, IP3R activation or IP3R suppression promoted or suppressed, respectively, Cx43 phosphorylation on S279/282. Site-directed mutagenesis indicated that expression of a mutant Cx43-S282A (alanine) inhibited S279/282 phosphorylation and GJ permeability, while the S279A mutant showed the opposite effect in ventricular myocytes. Expression of these mutants in HEK293 cells revealed that cells with a dual S279/282 mutation failed to express exogenous Cx43, whereas cells with a single S279 or S282 mutation displayed Cx43 overexpression with increased phosphorylation of S279/282 and promotion of intercellular communication.

Conclusions: These results demonstrated, for the first time, that IP3R physically interacts with Cx43 and participates in the regulation of Cx43 phosphorylation on S279/282, thereby affecting GJ intercellular communication in ventricular myocytes.

Show MeSH
Related in: MedlinePlus