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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-GFPY242F 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-GFPY242F 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-GFPY242F 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-GFPY242F transfected cells
recording in the presence of 100 µM of bpV(Phen) (n=3). (C)
Myoblasts expressing Kir2.1-GFP (control), Kir2.1-GFPY336F (Y336F),
Kir2.1-GFPY366F (Y366F) and Kir2.1-GFPY242F (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).
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