Fig. 4. Kir2.1 channels are modulated through the phosphorylation of the tyrosine 242. (A) Myoblasts expressing Kir2.1-GFP were incubated either with 10 µM bpV(Phen) for 6 hours or with 10 µM genistein for 4 hours. Myoblasts transfected with a vector containing only GFP was included in these experiments as a control. In all conditions, myoblasts were treated for 30 minutes before lysis with 10 µM bpV(Phen) to avoid unspecific dephosphorylation (this does not affect Kir2.1 current, data not shown). Kir2.1-GFP channels were immunoprecipitated from cell lysates with an anti-Kir2.1 antibody. Immunoprecipitated proteins were separated on SDS-PAGE, and revealed first with an anti-phosphotyrosine antibody (lower lane) and then reblotted with an anti-Kir2.1 antibody (upper lane). IP is for immunoprecipitation and IB for immunoblot. Bands were quantified by Optiquant software and represented as a histogram. The fraction of Kir2.1-GFP channels that are tyrosine-phosphorylated was normalized to the maximum tyrosine-phosphorylated Kir2.1-GFP channels obtained in the presence of 10 µM bpV(Phen). Results were obtained from three independent experiments. (B) Kir2.1-GFP and Kir2.1-GFP_Y242F currents were recorded in transfected proliferating myoblasts with a patch pipette containing or not (control) 100 µM bpV(Phen). Top: examples of Kir2.1-GFP and Kir2.1-GFP_Y242F currents recorded in the presence of 100 µM bpV(Phen) during voltage-steps as in Fig. 3A. Addition of Ba²⁺ (500 µM) blocked the Kir2.1-GFP_Y242F current at the end of the 25 minute recording. Bottom: Kir2.1 current amplitude was assessed as in Fig. 3B. The graph represents the mean Kir2.1 current of Kir2.1-GFP transfected cells for 25 minutes recording in the absence (n=5) or in the presence (n=3) of 100 µM of bpV(Phen), and of Kir2.1-GFP_Y242F transfected cells recording in the presence of 100 µM of bpV(Phen) (n=3). (C) Myoblasts expressing Kir2.1-GFP (control), Kir2.1-GFP_Y336F (Y336F), Kir2.1-GFP_Y366F (Y366F) and Kir2.1-GFP_Y242F (Y242F) channels were incubated with 200 µM bpV(Phen) for 1 hour. The Kir2.1 current density was evaluated before (gray) and after bpV(Phen) treatment by whole-cell patch-clamp. Nine to 16 cells were recorded in each condition; capacitances of each group of cells vary between 10±2 and 14±2 pF (not significantly different).