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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.
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