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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

IP3R-associated regulation of Cx43 phosphorylation in ventricular cardiomyocytes. Solubilized lysates from homogenized NRVMs (A) and mouse ventricles (C) that had been treated with different reagents as indicated were subjected to specific anti-phosphor Cx43 and anti-Cx43 antibodies, respectively. Representative western blots of pS368, pS262, pS279/282 and Cx43 as indicated (A) and their normalized relative abundances in NRVMs (B), and the blots of pS279/282 and Cx43 (C) and their normalized data in adult ventricles (D) depict the effects of 2-APB, shRNA against pan-IP3R, IP3/BM and ATP at the concentrations similar to those in functional evaluation on Cx43 phosphorylation. *P <0.05 and **P < 0.01 vs. DMSO- or scramble shRNA-treated cells, n = 3–4 independent experiments for each bar.
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Fig4: IP3R-associated regulation of Cx43 phosphorylation in ventricular cardiomyocytes. Solubilized lysates from homogenized NRVMs (A) and mouse ventricles (C) that had been treated with different reagents as indicated were subjected to specific anti-phosphor Cx43 and anti-Cx43 antibodies, respectively. Representative western blots of pS368, pS262, pS279/282 and Cx43 as indicated (A) and their normalized relative abundances in NRVMs (B), and the blots of pS279/282 and Cx43 (C) and their normalized data in adult ventricles (D) depict the effects of 2-APB, shRNA against pan-IP3R, IP3/BM and ATP at the concentrations similar to those in functional evaluation on Cx43 phosphorylation. *P <0.05 and **P < 0.01 vs. DMSO- or scramble shRNA-treated cells, n = 3–4 independent experiments for each bar.

Mentions: Compared with other connexins, Cx43 has a larger intracellular C-terminal tail, and (de)phosphorylation of serines in this domain represents an important regulatory mechanism for GJ gating, assembly, trafficking, and degradation. To investigate whether IP3/IP3R pathway affects GJ permeability by mediating the Cx43 phosphorylation, the relative abundance of pS262, pS368, and pS279/282 was analyzed by western blot analysis using phospho-specific antibodies. While one band of about 43 kDa appeared with the pS262 or pS368 antibody labeling, two clear bands were detected with the pS279/282 antibody labeling of samples from both NRVMs and adult ventricles (Figure 4A). Activation of PKC with PMA caused a significant increase in S368 phosphorylation and a slight increase in S279/282 phosphorylation, which is consistent with previous reports [10,12]. However, interestingly, inhibition of IP3R with 2-APB (3 μM) or shRNA against pan-IP3R significantly suppressed the levels of S279/282 phosphorylation, while no significant change was observed in S262 or S368 phosphorylation (Figure 4A and B). Furthermore, activation of IP3R with IP3/BM (20 μM) or ATP (5 μM) promoted Cx43 phosphorylation at S279/282 in NRVMs, and similar results were obtained in adult ventricles that were retrogradely perfused in a Langendorff system with HEPES-buffered Tyrode solution containing 2-APB, XeC, or ATP for 10, 20, or 5 minutes, respectively (Figure 4C and D).Figure 4


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)

IP3R-associated regulation of Cx43 phosphorylation in ventricular cardiomyocytes. Solubilized lysates from homogenized NRVMs (A) and mouse ventricles (C) that had been treated with different reagents as indicated were subjected to specific anti-phosphor Cx43 and anti-Cx43 antibodies, respectively. Representative western blots of pS368, pS262, pS279/282 and Cx43 as indicated (A) and their normalized relative abundances in NRVMs (B), and the blots of pS279/282 and Cx43 (C) and their normalized data in adult ventricles (D) depict the effects of 2-APB, shRNA against pan-IP3R, IP3/BM and ATP at the concentrations similar to those in functional evaluation on Cx43 phosphorylation. *P <0.05 and **P < 0.01 vs. DMSO- or scramble shRNA-treated cells, n = 3–4 independent experiments for each bar.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4195880&req=5

Fig4: IP3R-associated regulation of Cx43 phosphorylation in ventricular cardiomyocytes. Solubilized lysates from homogenized NRVMs (A) and mouse ventricles (C) that had been treated with different reagents as indicated were subjected to specific anti-phosphor Cx43 and anti-Cx43 antibodies, respectively. Representative western blots of pS368, pS262, pS279/282 and Cx43 as indicated (A) and their normalized relative abundances in NRVMs (B), and the blots of pS279/282 and Cx43 (C) and their normalized data in adult ventricles (D) depict the effects of 2-APB, shRNA against pan-IP3R, IP3/BM and ATP at the concentrations similar to those in functional evaluation on Cx43 phosphorylation. *P <0.05 and **P < 0.01 vs. DMSO- or scramble shRNA-treated cells, n = 3–4 independent experiments for each bar.
Mentions: Compared with other connexins, Cx43 has a larger intracellular C-terminal tail, and (de)phosphorylation of serines in this domain represents an important regulatory mechanism for GJ gating, assembly, trafficking, and degradation. To investigate whether IP3/IP3R pathway affects GJ permeability by mediating the Cx43 phosphorylation, the relative abundance of pS262, pS368, and pS279/282 was analyzed by western blot analysis using phospho-specific antibodies. While one band of about 43 kDa appeared with the pS262 or pS368 antibody labeling, two clear bands were detected with the pS279/282 antibody labeling of samples from both NRVMs and adult ventricles (Figure 4A). Activation of PKC with PMA caused a significant increase in S368 phosphorylation and a slight increase in S279/282 phosphorylation, which is consistent with previous reports [10,12]. However, interestingly, inhibition of IP3R with 2-APB (3 μM) or shRNA against pan-IP3R significantly suppressed the levels of S279/282 phosphorylation, while no significant change was observed in S262 or S368 phosphorylation (Figure 4A and B). Furthermore, activation of IP3R with IP3/BM (20 μM) or ATP (5 μM) promoted Cx43 phosphorylation at S279/282 in NRVMs, and similar results were obtained in adult ventricles that were retrogradely perfused in a Langendorff system with HEPES-buffered Tyrode solution containing 2-APB, XeC, or ATP for 10, 20, or 5 minutes, respectively (Figure 4C and D).Figure 4

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