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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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Related in: MedlinePlus

Co-localization of IP3R with Cx43 in gap junctions of ventricular myocytes. Neonatal rat (A) and adult mouse ventricle tissues (B), and cultured NRVMs (C) were co-immunostained with anti-Cx43 and anti-pan-IP3R antibodies. Representative confocal images show the subcellular distributions of Cx43 (red), pan-IP3R (green) and their co-localization (yellow) in the interfaces between adjacent myocytes. Three-dimensional reconstructions of a single disc of the end-to-end cells display that IP3R partially co-localized with Cx43 in GJs in ventricles (bottom panels in A and B). The enlarged interfaces 1 and 2 (inset in C) show the overlapped distribution of IP3R and Cx43 in NRVMs. Nucleus was stained with Hoechst 33258 (1 μg/ml). Scale bar: 10 μm. The yellow and white arrows in all panels indicate the enhanced IP3R signal in ventricles, and a fraction of Cx43 that is not associated with IP3R in GJs of connected NRVMs, respectively. Solubilized lysates from homogenized NRVMs (D) and mouse ventricles (E) were subjected to immunoprecipitation with anti-Cx43, anti-pan-IP3R, or anti-IP3R isoform antibodies as indicated. Representative western blots show the co-immunoprecipitated Cx43 or IP3R probed with anti-Cx43 or anti-pan-IP3R antibody. Data in all panels are representatives of 3–5 independent experiments.
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Fig1: Co-localization of IP3R with Cx43 in gap junctions of ventricular myocytes. Neonatal rat (A) and adult mouse ventricle tissues (B), and cultured NRVMs (C) were co-immunostained with anti-Cx43 and anti-pan-IP3R antibodies. Representative confocal images show the subcellular distributions of Cx43 (red), pan-IP3R (green) and their co-localization (yellow) in the interfaces between adjacent myocytes. Three-dimensional reconstructions of a single disc of the end-to-end cells display that IP3R partially co-localized with Cx43 in GJs in ventricles (bottom panels in A and B). The enlarged interfaces 1 and 2 (inset in C) show the overlapped distribution of IP3R and Cx43 in NRVMs. Nucleus was stained with Hoechst 33258 (1 μg/ml). Scale bar: 10 μm. The yellow and white arrows in all panels indicate the enhanced IP3R signal in ventricles, and a fraction of Cx43 that is not associated with IP3R in GJs of connected NRVMs, respectively. Solubilized lysates from homogenized NRVMs (D) and mouse ventricles (E) were subjected to immunoprecipitation with anti-Cx43, anti-pan-IP3R, or anti-IP3R isoform antibodies as indicated. Representative western blots show the co-immunoprecipitated Cx43 or IP3R probed with anti-Cx43 or anti-pan-IP3R antibody. Data in all panels are representatives of 3–5 independent experiments.

Mentions: To detect the three IP3R isoforms that are expressed in neonatal and adult ventricles [16-18], anti-pan-IP3R antibodies and anti-Cx43 antibodies were used to co-immunolabel samples. Figure 1A and B show representative interface region (upper panels) and three-dimensional reconstructions of the end-to-end intercalated discs between paired adjoining cardiomyocytes (bottom panels). Obvious co-localization of IP3R and Cx43 in GJ plaques (yellow color in upper panels) was observed in neonatal rat (Figure 1A) and adult mouse ventricular tissues (Figure 1B: here we used adult mice, because the IP3R distribution at the discs has already been reported in adult rat ventricles [20]). The front face disc view of GJ complexes demonstrated that the neonatal ventricular discs were fewer and smaller in size than those in adult discs, and the IP3R partially co-localized with Cx43, in particular in the larger GJs in both neonatal and adult ventricles (bottom panels). To conveniently expressing exogenous Cx43, NRVMs were used and co-immunostained with anti-pan-IP3R and anti-Cx43 antibodies. Similar to the neonatal tissue samples, IP3R clearly co-localized with Cx43, but there was still a small fraction of Cx43 that was not associated with IP3R in the GJ plaques of tissues and NRVMs (indicated with white arrows in Figure 1A and C). Additionally, it appears that there was more co-distribution of the two proteins in the total GJs of the neonatal samples than those in the adult samples, a difference likely due to a reduced IP3R expression after maturation [17-19].Figure 1


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)

Co-localization of IP3R with Cx43 in gap junctions of ventricular myocytes. Neonatal rat (A) and adult mouse ventricle tissues (B), and cultured NRVMs (C) were co-immunostained with anti-Cx43 and anti-pan-IP3R antibodies. Representative confocal images show the subcellular distributions of Cx43 (red), pan-IP3R (green) and their co-localization (yellow) in the interfaces between adjacent myocytes. Three-dimensional reconstructions of a single disc of the end-to-end cells display that IP3R partially co-localized with Cx43 in GJs in ventricles (bottom panels in A and B). The enlarged interfaces 1 and 2 (inset in C) show the overlapped distribution of IP3R and Cx43 in NRVMs. Nucleus was stained with Hoechst 33258 (1 μg/ml). Scale bar: 10 μm. The yellow and white arrows in all panels indicate the enhanced IP3R signal in ventricles, and a fraction of Cx43 that is not associated with IP3R in GJs of connected NRVMs, respectively. Solubilized lysates from homogenized NRVMs (D) and mouse ventricles (E) were subjected to immunoprecipitation with anti-Cx43, anti-pan-IP3R, or anti-IP3R isoform antibodies as indicated. Representative western blots show the co-immunoprecipitated Cx43 or IP3R probed with anti-Cx43 or anti-pan-IP3R antibody. Data in all panels are representatives of 3–5 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: Co-localization of IP3R with Cx43 in gap junctions of ventricular myocytes. Neonatal rat (A) and adult mouse ventricle tissues (B), and cultured NRVMs (C) were co-immunostained with anti-Cx43 and anti-pan-IP3R antibodies. Representative confocal images show the subcellular distributions of Cx43 (red), pan-IP3R (green) and their co-localization (yellow) in the interfaces between adjacent myocytes. Three-dimensional reconstructions of a single disc of the end-to-end cells display that IP3R partially co-localized with Cx43 in GJs in ventricles (bottom panels in A and B). The enlarged interfaces 1 and 2 (inset in C) show the overlapped distribution of IP3R and Cx43 in NRVMs. Nucleus was stained with Hoechst 33258 (1 μg/ml). Scale bar: 10 μm. The yellow and white arrows in all panels indicate the enhanced IP3R signal in ventricles, and a fraction of Cx43 that is not associated with IP3R in GJs of connected NRVMs, respectively. Solubilized lysates from homogenized NRVMs (D) and mouse ventricles (E) were subjected to immunoprecipitation with anti-Cx43, anti-pan-IP3R, or anti-IP3R isoform antibodies as indicated. Representative western blots show the co-immunoprecipitated Cx43 or IP3R probed with anti-Cx43 or anti-pan-IP3R antibody. Data in all panels are representatives of 3–5 independent experiments.
Mentions: To detect the three IP3R isoforms that are expressed in neonatal and adult ventricles [16-18], anti-pan-IP3R antibodies and anti-Cx43 antibodies were used to co-immunolabel samples. Figure 1A and B show representative interface region (upper panels) and three-dimensional reconstructions of the end-to-end intercalated discs between paired adjoining cardiomyocytes (bottom panels). Obvious co-localization of IP3R and Cx43 in GJ plaques (yellow color in upper panels) was observed in neonatal rat (Figure 1A) and adult mouse ventricular tissues (Figure 1B: here we used adult mice, because the IP3R distribution at the discs has already been reported in adult rat ventricles [20]). The front face disc view of GJ complexes demonstrated that the neonatal ventricular discs were fewer and smaller in size than those in adult discs, and the IP3R partially co-localized with Cx43, in particular in the larger GJs in both neonatal and adult ventricles (bottom panels). To conveniently expressing exogenous Cx43, NRVMs were used and co-immunostained with anti-pan-IP3R and anti-Cx43 antibodies. Similar to the neonatal tissue samples, IP3R clearly co-localized with Cx43, but there was still a small fraction of Cx43 that was not associated with IP3R in the GJ plaques of tissues and NRVMs (indicated with white arrows in Figure 1A and C). Additionally, it appears that there was more co-distribution of the two proteins in the total GJs of the neonatal samples than those in the adult samples, a difference likely due to a reduced IP3R expression after maturation [17-19].Figure 1

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