Fig. 5. Kir2.1-GFP channels are not internalized by the bpV(Phen)-treatment. (A) Myoblasts expressing Kir2.1-GFP channels were incubated with or without 10 µM bpV(Phen) for 6 hours. After cell surface protein biotinylation, myoblasts were lysed and an equal amount of proteins from each condition was precipitated using streptavidin magnetic particles. Myoblasts expressing Kir2.1-GFP channels on which no biotinylation was performed and myoblasts transfected with a vector containing only GFP were included as controls. Total proteins and biotinylated proteins were separated on SDS-PAGE and revealed with an anti-Kir2.1 antibody. Bands were quantified by Optiquant software. The histogram represents the fraction of biotinylated Kir2.1-GFP channels normalized to the fraction of biotinylated Kir2.1-GFP channels observed in the absence of bpV(Phen). Results were obtained from four independent experiments. (B) Myoblasts expressing Kir2.1-GFP channels were incubated with 200 µM bpV(Phen) for 1 hour. The Kir2.1-GFP current density (gray) was evaluated before (n=3) and after (n=5) bpV(Phen) treatment by whole-cell patch-clamp technique. In parallel experiments, Kir2.1-GFP channels fluorescence (black) was followed by TIRF microscopy for the 1 hour bpV(Phen) treatment. The histogram represents Kir2.1-GFP current density before and after bpV(Phen) treatment and the average fluorescence from four myoblasts (mean of 7-8 regions per cell) measured during the first 3 and the final 3 minutes of the 1 hour bpV(Phen) treatment. The fluorescence was normalized to the fluorescence measured during the first 3 minutes of the bpV(Phen) treatment. TIRF pictures of a myoblast at the beginning and at the end of the 1 hour bpV(Phen) treatment are shown on the right.