Intercellular communication mediated by Cx43 triple map kinase consensus site mutant: Single channel analysis, EGF response

Epidermal growth factor (EGF) has been demonstrated to activate the Ras/Raf/MEK/MAP kinase cascade. Previous studies have suggested that the connexin (Cx) 43 gap junction protein is phosphorylated by MAP kinase and that EGF treatment of cells expressing Cx43 results in diminished intercellular communication. MAP kinase consensus sequences have been identified in the C-terminus of Cx43 with serines 255, 279, and 282 being recognized as targets for phosphorylation. The purpose of this study was to investigate whether EGF mediated decreases in intercellular communication could be attenuated by serine to alanine mutations (S255,279,282A) on Cx43. Mouse fibroblast Cx43 knockouts were transfected with either wild type Cx43 (Cx43-wt, C9 cells) or a Cx43 triple mutant (Cx43-S255,279,282A, G11 cells) (both cell types were kindly provided by Warn-Cramer and Lau). Cells were cultured to confluency and replated on coverslips at low density. Cell pairs were isolated and a dual whole-cell voltage clamp protocol was used to record macroscopic junctional conductance (g_j) and single channel conductance in the presence and absence of EGF (100ng/ml). Mean g_j in the C9, Cx43-wt expressing cells, was 22.4 ± 2.8nS, n=14. Comparable levels of coupling were observed in the Cx43-S255,279,282A mutant expressing cells, 18.9 ± 2.0nS, n=14. EGF induced a ∼50% decrease in g_j within minutes in the Cx43-wt expressing cells but had little effect in the Cx43-S255,279,282A mutant expressing cells. Single channel analysis revealed that unitary conductance was comparable between C9 and G11 cells, with 60 and 90pS channels being most prominent. Preliminary findings indicate that the relative frequency of smaller channels are increased in both cell types in response to EGF. Despite the absence of a change in g_j, for the G11 mutant cells, relative frequency histograms indicate EGF induced changes at the single channel level. The functional ramifications of these observations remain to be elucidated.