Phosphorylation of connexin43 on serine 306 regulates electrical coupling.
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.


CONCLUSIONS
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.
Procida
Kristina
K
Danish National Research Foundation Centre for Cardiac Arrhythmia at Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark.
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
D002450:Cell Communication / Q000502:physiology*; D018031:Connexin 43 / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism*
D018031:Connexin 43; D012694:Serine