Phosphorylation of connexin43 on serine 306 regulates electrical coupling

• Published in: Heart rhythm Vol. 6; no. 11; pp. 1632 - 1638
• Main Authors: Procida, Kristina, MD, Jørgensen, Lone, MS, Schmitt, Nicole, PhD, Delmar, Mario, MD, PhD, FHRS, Taffet, Steven M., PhD, Holstein-Rathlou, Niels-Henrik, MD, PhD, Nielsen, Morten Schak, PhD, Braunstein, Thomas Hartig, PhD
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-11-2009
• Subjects: Cardiovascular; Cell Communication - physiology; Connexin 43 - metabolism; Connexin43; Gap junction; Humans; Ischemia; Phosphorylation; Serine - metabolism; Single channel; Single channel; PBS; Ab; Phosphorylation; antibody; phenylmethanesulfonyl fluoride; PKC; Gap junction; BSA; Ischemia; Cx43; PMSF; phosphate buffered saline; Connexin43; wild type; WT; bovine serum albumin; protein kinase C
• ISSN: 1547-5271; 1556-3871
• DOI: 10.1016/j.hrthm.2009.07.043

Background 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. Objective The purpose of this study was to examine the role of S296, S297, and S306 in the regulation of electrical intercellular communication. Methods 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. Results 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. Conclusion 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.