The Experts below are selected from a list of 34965 Experts worldwide ranked by ideXlab platform
Almke Bader - One of the best experts on this subject based on the ideXlab platform.
-
adenosine receptors regulate Gap Junction coupling of the human cerebral microvascular endothelial cells hcmec d3 by ca2 influx through cyclic nucleotide gated channels
The Journal of Physiology, 2017Co-Authors: Almke Bader, Daniela Begandt, Willem Bintig, Anne Klett, Ina G Siller, Carola Gregor, Frank Schaarschmidt, Babette B Weksler, Ignacio A Romero, Pierreolivier CouraudAbstract:The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the Gap Junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A(2A) and A(2B) adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the Gap Junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced Gap Junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase in Gap Junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of Gap Junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor l-cis-diltiazem, as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+, suppressed the 2-PAA-related enhancement of Gap Junction coupling. Moreover, we observed a 2-PAA-dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP-dependent manner. Ca2+ in turn induces the formation of new Gap Junction plaques and a consecutive sustained enhancement of Gap Junction coupling. The report identifies CNG channels as a physiological link that integrates Gap Junction coupling into the adenosine receptor-dependent signalling of endothelial cells of the blood-brain barrier.
-
adenosine receptors regulate Gap Junction coupling of the human cerebral microvascular endothelial cells hcmec d3 by ca2 influx through cng channels
The Journal of Physiology, 2017Co-Authors: Almke Bader, Daniela Begandt, Willem Bintig, Anne Klett, Ina G Siller, Carola Gregor, Frank Schaarschmidt, Babette B Weksler, Ignacio A Romero, Pierreolivier CouraudAbstract:The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the Gap Junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A2A and A2B adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the Gap Junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced Gap Junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase of Gap Junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of Gap Junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor L-cis-diltiazem as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+ suppressed the 2-PAA-related enhancement of Gap Junction coupling. Moreover, we observed a 2-PAA dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP-dependent manner. Ca2+ in turn induces the formation of new Gap Junction plaques and a consecutive sustained enhancement of Gap Junction coupling. The report identifies CNG channels as a physiological link that integrates Gap Junction coupling into the adenosine receptor dependent signalling of endothelial cells of the blood-brain barrier. This article is protected by copyright. All rights reserved
-
dipyridamole related enhancement of Gap Junction coupling in the gm 7373 aortic endothelial cells correlates with an increase in the amount of connexin 43 mrna and protein as well as Gap Junction plaques
Journal of Bioenergetics and Biomembranes, 2013Co-Authors: Daniela Begandt, Almke Bader, Lutz Dreyer, Anaclet Ngezahayo, Linda Gerhard, Julia Lindner, Barbara SchlingmannAbstract:Previous data showed that dipyridamole enhanced Gap Junction coupling in vascular endothelial and smooth muscle cell lines by a cAMP-dependent mechanism. The present study investigates the level at which dipyridamole affects Gap Junction coupling. In the GM-7373 endothelial cell line, scrape loading/dye transfer experiments revealed a rapid increase in Gap Junction coupling induced during the first 6 h of dipyridamole treatment, followed by a slow increase induced by further incubation. Immunostaining analyses showed that the rapid enhancement of Gap Junction coupling correlated with an increased amount of Cx43 Gap Junction plaques and a reduced amount of Cx43 containing vesicles, while the amount of Cx43 mRNA or protein was not changed during this period, as found by semiquantitative RT-PCR and Western blot. Additionally, brefeldin A did not block this short-term-induced enhancement of Gap Junction coupling. Along with the dipyridamole-induced long-term enhancement of Gap Junction coupling, the amount of Cx43 mRNA and protein additionally to the amount of Cx43 Gap Junction plaques were increased. Furthermore, the anti-Cx43 antibody detected only two bands at 42 kDa and 44 kDa in control cells and cells treated with dipyridamole for 6 h, while long-term dipyridamole-treated cells showed a third band at 46 kDa. We propose that a dipyridamole-induced cAMP synthesis increased Gap Junction coupling in the GM-7373 endothelial cell line at different levels: the short-term effect is related to already oligomerised connexins beyond the Golgi apparatus and the long-term effect involves new expression and synthesis as well as posttranslational modification of Cx43.
-
Biphasic increase of Gap Junction coupling induced by dipyridamole in the rat aortic A-10 vascular smooth muscle cell line
Journal of Cell Communication and Signaling, 2013Co-Authors: Daniela Begandt, Almke Bader, Lutz Dreyer, Natalie Eisert, Thilo Reeck, Anaclet NgezahayoAbstract:The rat aortic smooth muscle cell line A-10 was used to investigate the effect of dipyridamole on the Gap Junction coupling of smooth muscle cells. The scrape loading/dye transfer (SL/DT) technique revealed that dipyridamole concentrations between 5 μM and 100 μM significantly increased Gap Junction coupling. The adenosine receptor antagonist MRS 1754, as well as the PKA inhibitors Rp-cAMPS and H-89 were able to inhibit the dipyridamole-related increase in coupling, while forskolin and Br-cAMP also induced an enhancement of the Gap Junction coupling. Regarding the time-dependent behaviour of dipyridamole, a short-term effect characterised by an oscillatory reaction was observed for application times of less than 5 h, while applications times of at least 6 h resulted in a long-term effect, characterised by a constant increase of Gap Junction coupling to its maximum levels. This increase was not altered by prolonged presence of dipyridamole. In parallel, a short application of dipyridamole for at least 15 min was found to be sufficient to evoke the long-term effect measured 6 h after drug washout. We propose that in both the short-term and long-term effect, cAMP-related pathways are activated. The short-term phase could be related to an oscillatory cAMP effect, which might directly affect connexin trafficking, assembly and/or Gap Junction gating. The long-term effect is most likely related to the new expression and synthesis of connexins. With previous data from a bovine aortic endothelial cell line, the present results show that Gap Junction coupling of vascular cells is a target for dipyridamole.
Daniela Begandt - One of the best experts on this subject based on the ideXlab platform.
-
adenosine receptors regulate Gap Junction coupling of the human cerebral microvascular endothelial cells hcmec d3 by ca2 influx through cyclic nucleotide gated channels
The Journal of Physiology, 2017Co-Authors: Almke Bader, Daniela Begandt, Willem Bintig, Anne Klett, Ina G Siller, Carola Gregor, Frank Schaarschmidt, Babette B Weksler, Ignacio A Romero, Pierreolivier CouraudAbstract:The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the Gap Junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A(2A) and A(2B) adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the Gap Junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced Gap Junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase in Gap Junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of Gap Junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor l-cis-diltiazem, as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+, suppressed the 2-PAA-related enhancement of Gap Junction coupling. Moreover, we observed a 2-PAA-dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP-dependent manner. Ca2+ in turn induces the formation of new Gap Junction plaques and a consecutive sustained enhancement of Gap Junction coupling. The report identifies CNG channels as a physiological link that integrates Gap Junction coupling into the adenosine receptor-dependent signalling of endothelial cells of the blood-brain barrier.
-
adenosine receptors regulate Gap Junction coupling of the human cerebral microvascular endothelial cells hcmec d3 by ca2 influx through cng channels
The Journal of Physiology, 2017Co-Authors: Almke Bader, Daniela Begandt, Willem Bintig, Anne Klett, Ina G Siller, Carola Gregor, Frank Schaarschmidt, Babette B Weksler, Ignacio A Romero, Pierreolivier CouraudAbstract:The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the Gap Junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A2A and A2B adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the Gap Junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced Gap Junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase of Gap Junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of Gap Junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor L-cis-diltiazem as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+ suppressed the 2-PAA-related enhancement of Gap Junction coupling. Moreover, we observed a 2-PAA dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP-dependent manner. Ca2+ in turn induces the formation of new Gap Junction plaques and a consecutive sustained enhancement of Gap Junction coupling. The report identifies CNG channels as a physiological link that integrates Gap Junction coupling into the adenosine receptor dependent signalling of endothelial cells of the blood-brain barrier. This article is protected by copyright. All rights reserved
-
dipyridamole related enhancement of Gap Junction coupling in the gm 7373 aortic endothelial cells correlates with an increase in the amount of connexin 43 mrna and protein as well as Gap Junction plaques
Journal of Bioenergetics and Biomembranes, 2013Co-Authors: Daniela Begandt, Almke Bader, Lutz Dreyer, Anaclet Ngezahayo, Linda Gerhard, Julia Lindner, Barbara SchlingmannAbstract:Previous data showed that dipyridamole enhanced Gap Junction coupling in vascular endothelial and smooth muscle cell lines by a cAMP-dependent mechanism. The present study investigates the level at which dipyridamole affects Gap Junction coupling. In the GM-7373 endothelial cell line, scrape loading/dye transfer experiments revealed a rapid increase in Gap Junction coupling induced during the first 6 h of dipyridamole treatment, followed by a slow increase induced by further incubation. Immunostaining analyses showed that the rapid enhancement of Gap Junction coupling correlated with an increased amount of Cx43 Gap Junction plaques and a reduced amount of Cx43 containing vesicles, while the amount of Cx43 mRNA or protein was not changed during this period, as found by semiquantitative RT-PCR and Western blot. Additionally, brefeldin A did not block this short-term-induced enhancement of Gap Junction coupling. Along with the dipyridamole-induced long-term enhancement of Gap Junction coupling, the amount of Cx43 mRNA and protein additionally to the amount of Cx43 Gap Junction plaques were increased. Furthermore, the anti-Cx43 antibody detected only two bands at 42 kDa and 44 kDa in control cells and cells treated with dipyridamole for 6 h, while long-term dipyridamole-treated cells showed a third band at 46 kDa. We propose that a dipyridamole-induced cAMP synthesis increased Gap Junction coupling in the GM-7373 endothelial cell line at different levels: the short-term effect is related to already oligomerised connexins beyond the Golgi apparatus and the long-term effect involves new expression and synthesis as well as posttranslational modification of Cx43.
-
Biphasic increase of Gap Junction coupling induced by dipyridamole in the rat aortic A-10 vascular smooth muscle cell line
Journal of Cell Communication and Signaling, 2013Co-Authors: Daniela Begandt, Almke Bader, Lutz Dreyer, Natalie Eisert, Thilo Reeck, Anaclet NgezahayoAbstract:The rat aortic smooth muscle cell line A-10 was used to investigate the effect of dipyridamole on the Gap Junction coupling of smooth muscle cells. The scrape loading/dye transfer (SL/DT) technique revealed that dipyridamole concentrations between 5 μM and 100 μM significantly increased Gap Junction coupling. The adenosine receptor antagonist MRS 1754, as well as the PKA inhibitors Rp-cAMPS and H-89 were able to inhibit the dipyridamole-related increase in coupling, while forskolin and Br-cAMP also induced an enhancement of the Gap Junction coupling. Regarding the time-dependent behaviour of dipyridamole, a short-term effect characterised by an oscillatory reaction was observed for application times of less than 5 h, while applications times of at least 6 h resulted in a long-term effect, characterised by a constant increase of Gap Junction coupling to its maximum levels. This increase was not altered by prolonged presence of dipyridamole. In parallel, a short application of dipyridamole for at least 15 min was found to be sufficient to evoke the long-term effect measured 6 h after drug washout. We propose that in both the short-term and long-term effect, cAMP-related pathways are activated. The short-term phase could be related to an oscillatory cAMP effect, which might directly affect connexin trafficking, assembly and/or Gap Junction gating. The long-term effect is most likely related to the new expression and synthesis of connexins. With previous data from a bovine aortic endothelial cell line, the present results show that Gap Junction coupling of vascular cells is a target for dipyridamole.
Pierreolivier Couraud - One of the best experts on this subject based on the ideXlab platform.
-
adenosine receptors regulate Gap Junction coupling of the human cerebral microvascular endothelial cells hcmec d3 by ca2 influx through cyclic nucleotide gated channels
The Journal of Physiology, 2017Co-Authors: Almke Bader, Daniela Begandt, Willem Bintig, Anne Klett, Ina G Siller, Carola Gregor, Frank Schaarschmidt, Babette B Weksler, Ignacio A Romero, Pierreolivier CouraudAbstract:The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the Gap Junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A(2A) and A(2B) adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the Gap Junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced Gap Junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase in Gap Junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of Gap Junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor l-cis-diltiazem, as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+, suppressed the 2-PAA-related enhancement of Gap Junction coupling. Moreover, we observed a 2-PAA-dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP-dependent manner. Ca2+ in turn induces the formation of new Gap Junction plaques and a consecutive sustained enhancement of Gap Junction coupling. The report identifies CNG channels as a physiological link that integrates Gap Junction coupling into the adenosine receptor-dependent signalling of endothelial cells of the blood-brain barrier.
-
adenosine receptors regulate Gap Junction coupling of the human cerebral microvascular endothelial cells hcmec d3 by ca2 influx through cng channels
The Journal of Physiology, 2017Co-Authors: Almke Bader, Daniela Begandt, Willem Bintig, Anne Klett, Ina G Siller, Carola Gregor, Frank Schaarschmidt, Babette B Weksler, Ignacio A Romero, Pierreolivier CouraudAbstract:The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the Gap Junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A2A and A2B adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the Gap Junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced Gap Junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase of Gap Junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of Gap Junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor L-cis-diltiazem as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+ suppressed the 2-PAA-related enhancement of Gap Junction coupling. Moreover, we observed a 2-PAA dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP-dependent manner. Ca2+ in turn induces the formation of new Gap Junction plaques and a consecutive sustained enhancement of Gap Junction coupling. The report identifies CNG channels as a physiological link that integrates Gap Junction coupling into the adenosine receptor dependent signalling of endothelial cells of the blood-brain barrier. This article is protected by copyright. All rights reserved
Anaclet Ngezahayo - One of the best experts on this subject based on the ideXlab platform.
-
dipyridamole related enhancement of Gap Junction coupling in the gm 7373 aortic endothelial cells correlates with an increase in the amount of connexin 43 mrna and protein as well as Gap Junction plaques
Journal of Bioenergetics and Biomembranes, 2013Co-Authors: Daniela Begandt, Almke Bader, Lutz Dreyer, Anaclet Ngezahayo, Linda Gerhard, Julia Lindner, Barbara SchlingmannAbstract:Previous data showed that dipyridamole enhanced Gap Junction coupling in vascular endothelial and smooth muscle cell lines by a cAMP-dependent mechanism. The present study investigates the level at which dipyridamole affects Gap Junction coupling. In the GM-7373 endothelial cell line, scrape loading/dye transfer experiments revealed a rapid increase in Gap Junction coupling induced during the first 6 h of dipyridamole treatment, followed by a slow increase induced by further incubation. Immunostaining analyses showed that the rapid enhancement of Gap Junction coupling correlated with an increased amount of Cx43 Gap Junction plaques and a reduced amount of Cx43 containing vesicles, while the amount of Cx43 mRNA or protein was not changed during this period, as found by semiquantitative RT-PCR and Western blot. Additionally, brefeldin A did not block this short-term-induced enhancement of Gap Junction coupling. Along with the dipyridamole-induced long-term enhancement of Gap Junction coupling, the amount of Cx43 mRNA and protein additionally to the amount of Cx43 Gap Junction plaques were increased. Furthermore, the anti-Cx43 antibody detected only two bands at 42 kDa and 44 kDa in control cells and cells treated with dipyridamole for 6 h, while long-term dipyridamole-treated cells showed a third band at 46 kDa. We propose that a dipyridamole-induced cAMP synthesis increased Gap Junction coupling in the GM-7373 endothelial cell line at different levels: the short-term effect is related to already oligomerised connexins beyond the Golgi apparatus and the long-term effect involves new expression and synthesis as well as posttranslational modification of Cx43.
-
Biphasic increase of Gap Junction coupling induced by dipyridamole in the rat aortic A-10 vascular smooth muscle cell line
Journal of Cell Communication and Signaling, 2013Co-Authors: Daniela Begandt, Almke Bader, Lutz Dreyer, Natalie Eisert, Thilo Reeck, Anaclet NgezahayoAbstract:The rat aortic smooth muscle cell line A-10 was used to investigate the effect of dipyridamole on the Gap Junction coupling of smooth muscle cells. The scrape loading/dye transfer (SL/DT) technique revealed that dipyridamole concentrations between 5 μM and 100 μM significantly increased Gap Junction coupling. The adenosine receptor antagonist MRS 1754, as well as the PKA inhibitors Rp-cAMPS and H-89 were able to inhibit the dipyridamole-related increase in coupling, while forskolin and Br-cAMP also induced an enhancement of the Gap Junction coupling. Regarding the time-dependent behaviour of dipyridamole, a short-term effect characterised by an oscillatory reaction was observed for application times of less than 5 h, while applications times of at least 6 h resulted in a long-term effect, characterised by a constant increase of Gap Junction coupling to its maximum levels. This increase was not altered by prolonged presence of dipyridamole. In parallel, a short application of dipyridamole for at least 15 min was found to be sufficient to evoke the long-term effect measured 6 h after drug washout. We propose that in both the short-term and long-term effect, cAMP-related pathways are activated. The short-term phase could be related to an oscillatory cAMP effect, which might directly affect connexin trafficking, assembly and/or Gap Junction gating. The long-term effect is most likely related to the new expression and synthesis of connexins. With previous data from a bovine aortic endothelial cell line, the present results show that Gap Junction coupling of vascular cells is a target for dipyridamole.
Paul D Lampe - One of the best experts on this subject based on the ideXlab platform.
-
spatio temporal regulation of connexin43 phosphorylation and Gap Junction dynamics
Biochimica et Biophysica Acta, 2018Co-Authors: Joell L Solan, Paul D LampeAbstract:: Gap Junctions are specialized membrane domains containing tens to thousands of intercellular channels. These channels permit exchange of small molecules (<1000Da) including ions, amino acids, nucleotides, metabolites and secondary messengers (e.g., calcium, glucose, cAMP, cGMP, IP3) between cells. The common reductionist view of these structures is that they are composed entirely of integral membrane proteins encoded by the 21 member connexin human gene family. However, it is clear that the normal physiological function of this structure requires interaction and regulation by a variety of proteins, especially kinases. Phosphorylation is capable of directly modulating connexin channel function but the most dramatic effects on Gap Junction activity occur via the organization of the Gap Junction structures themselves. This is a direct result of the short half-life of the primary Gap Junction protein, connexin, which requires them to be constantly assembled, remodeled and turned over. The biological consequences of this remodeling are well illustrated during cardiac ischemia, a process wherein Gap Junctions are disassembled and remodeled resulting in arrhythmia and ultimately heart failure. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.
-
activation of akt not connexin 43 protein ubiquitination regulates Gap Junction stability
Journal of Biological Chemistry, 2012Co-Authors: Clarence A Dunn, Alan F Lau, Paul D LampeAbstract:The pore-forming Gap Junctional protein connexin 43 (Cx43) has a short (1–3 h) half-life in cells in tissue culture and in whole tissues. Although critical for cellular function in all tissues, the process of Gap Junction turnover is not well understood because treatment of cells with a proteasomal inhibitor results in larger Gap Junctions but little change in total Cx43 protein whereas lysosomal inhibitors increase total, mostly nonJunctional Cx43. To better understand turnover and identify potential sites of Cx43 ubiquitination, we prepared constructs of Cx43 with different lysines converted to arginines. However, when transfected into cells, a mutant version of Cx43 with all lysines converted to arginines behaved similarly to wild type in the presence of proteasomal and lysosomal inhibitors, indicating that ubiquitination of Cx43 did not appear to be playing a role in Gap Junction stability. Through the use of inhibitors and dominant negative constructs, we found that Akt (protein kinase B) activity controlled Gap Junction stability and was necessary to form larger stable Gap Junctions. Akt activation was increased upon proteasomal inhibition and resulted in phosphorylation of Cx43 at Akt phosphorylation consensus sites. Thus, we conclude that Cx43 ubiquitination is not necessary for the regulation of Cx43 turnover; rather, Akt activity, probably through direct phosphorylation of Cx43, controls Gap Junction stability. This linkage of a kinase involved in controlling cell survival and growth to Gap Junction stability may mechanistically explain how Gap Junctions and Akt play similar regulatory roles.
-
casein kinase 1 regulates connexin 43 Gap Junction assembly
Journal of Biological Chemistry, 2002Co-Authors: Cynthia D Cooper, Paul D LampeAbstract:Abstract Phosphorylation of members of the connexin family of Gap Junction proteins has been correlated with Gap Junction assembly, but the mechanisms involved remain unclear. We have examined the role of casein kinase 1 (CK1) in connexin-43 (Cx43) Gap Junction assembly. Cellular co-immunoprecipitation experiments and in vitro CK1 phosphorylation reactions indicate that CK1 interacted with and phosphorylated Cx43, initially on serine(s) 325, 328, or 330.32Pi-Metabolically labeled cells treated with CKI-7, a specific CK1 inhibitor, showed a reduction in Cx43 phosphorylation on site(s) that can be phosphorylated by CK1 in vitro. To examine CK1 function, normal rat kidney cells were treated with CKI-7, and Cx43 content was analyzed by Triton X-100 extraction, cell-surface biotinylation, and immunofluorescence. Western blot analysis indicated a slight increase in total Cx43, whereas Gap Junctional (Triton-insoluble) Cx43 decreased, and non-Junctional plasma membrane Cx43 increased (as detected by cell surface biotinylation). Immunofluorescence experiments in the presence of CK1 inhibitor showed increases in Cx43 plasma membrane localization but not necessarily accumulation at cell-cell interfaces. Decreased Gap Junctional and phosphorylated Cx43 was also detected when cells were treated with IC261, a CK1 inhibitor specific for δ or e isoforms. These data suggest CK1δ could regulate Cx43 Gap Junction assembly by directly phosphorylating Cx43.
