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Phosphorylation of connexin43 on serine 306 regulates electrical coupling.

ABSTRACT: Phosphorylation is a key regulatory event in controlling the function of the cardiac gap junction protein connexin43 (Cx43). Three new phosphorylation sites (S296, S297, S306) have been identified on Cx43; two of these sites (S297 and S306) are dephosphorylated during ischemia. The functional significance of these new sites is currently unknown.The purpose of this study was to examine the role of S296, S297, and S306 in the regulation of electrical intercellular communication.To mimic constitutive dephosphorylation, serine was mutated to alanine at the three sites and expressed in HeLa cells. Electrical coupling and single channel measurements were performed by double patch clamp. Protein expression levels were assayed by western blotting, localization of Cx43, and phosphorylation of S306 by immunolabeling. Free hemichannels were assessed by biotinylation.Macroscopic conductance in cells expressing S306A was reduced to 57% compared to wild type (WT), whereas coupling was not significantly changed in cells expressing either S296A or S297A. S306A-expressing cells displayed similar protein and free hemichannel abundance compared to WT Cx43, whereas the fractional area of plaques in cell-to-cell interfaces was increased. However, single channel measurements showed a WT Cx43 main state conductance of 119 pS, whereas the main state conductance of S306A channels was reduced to 95 pS. Furthermore, channel gating was affected in S306A channels.Lack of phosphorylation at serine 306 results in reduced coupling, which can be explained by reduced single channel conductance. We suggest that dephosphorylation of S306 partly explains the electrical uncoupling seen in myocardial ischemia.

SUBMITTER: Procida K

PROVIDER: S-EPMC2803062 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Phosphorylation of connexin43 on serine 306 regulates electrical coupling.

Procida Kristina K Jørgensen Lone L Schmitt Nicole N Delmar Mario M Taffet Steven M SM Holstein-Rathlou Niels-Henrik NH Nielsen Morten Schak MS Braunstein Thomas Hartig TH

Heart rhythm 20090728 11

<h4>Background</h4>Phosphorylation is a key regulatory event in controlling the function of the cardiac gap junction protein connexin43 (Cx43). Three new phosphorylation sites (S296, S297, S306) have been identified on Cx43; two of these sites (S297 and S306) are dephosphorylated during ischemia. The functional significance of these new sites is currently unknown.<h4>Objective</h4>The purpose of this study was to examine the role of S296, S297, and S306 in the regulation of electrical intercellu ...[more]

PMID: 19879542

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Project description:To investigate whether connexin phosphorylation regulates the known role of zonula occludens-1 protein (ZO-1) in gap junction (GJ) function, we generated and analyzed a series of phosphomimetic and phosphorylation-dead mutants by mutating known conserved regulatory serine (S) residues 255, 279/282, 365, 368, and 373 located in the C-terminal domain of connexin43 (Cx43) into glutamic acid (E) or alanine (A) residues. All connexin mutants were translated into stable, full-length proteins and assembled into GJs when expressed in HeLa or Madin-Darby canine kidney epithelial cells. However, mutants with S residues exchanged at positions 365, 368, and 373 exhibited a significantly altered ZO-1 interaction profile, while mutants with S residues exchanged at 255 and 279/282 did not. Unlike wild-type Cx43, in which ZO-1 binding is restricted to the periphery of GJ plaques, S365A, S365E, S368A, S368E, and S373A mutants bound ZO-1 throughout the GJ plaques, while the S373E mutant did not bind ZO-1 at all. Inability to disengage from ZO-1 correlated with increased GJ plaque size and increased connexin protein half-life, while maintaining GJ channels in an open, functional state. Quantitative clathrin-binding analyses revealed no significant alterations in clathrin-binding efficiency, suggesting that the inability to disengage from ZO-1 prevented maturation of functional into nonfunctional/endocytic channels, rather than ZO-1 interfering with GJ endocytosis directly. Collectively, our results indicate that ZO-1 binding regulates channel accrual, while disengagement from ZO-1 is critical for GJ channel closure and transitioning GJ channels for endocytosis. Intriguingly, these transitional ZO-1 binding/release and channel-aging steps are mediated by a series of hierarchical phosphorylation/dephosphorylation events at S373, S365, and S368, well-known Cx43 Akt, protein kinase A, and protein kinase C phosphorylation sites located in the vicinity of the ZO-1 binding site.

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Phosphorylation of connexin43 on serine 306 regulates electrical coupling.

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Phosphorylation of connexin43 on serine 306 regulates electrical coupling.

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