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First published online 23 January 2008
doi: 10.1242/dev.011387
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1 Département de Neurosciences Fondamentales, University of Geneva,
Centre Médical Universitaire, CH-1211 Geneva 4, Switzerland.
2 Département de Pathologie et Immunologie, University of Geneva, Centre
Médical Universitaire, CH-1211 Geneva 4, Switzerland.
3 Département des Neurosciences Cliniques, University of Geneva, Centre
Médical Universitaire, CH-1211 Geneva 4, Switzerland.
* Author for correspondence (e-mail: laurent.bernheim{at}medecine.unige.ch)
Accepted 18 December 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Human myoblasts, Hyperpolarization, Myoblast differentiation, Potassium channel, Tyrosine phosphorylation
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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We have previously shown that human myoblasts must hyperpolarize before
they can differentiate (Liu et al.,
1998
), and that this hyperpolarization occurs via an increased
activity of Kir2.1 K+ channels
(Fischer-Lougheed et al.,
2001). Hyperpolarization of myoblasts induces a Ca2+
influx that is an essential early step of the differentiation process
(Bijlenga et al., 2000). We
have also established that Kir2.1 channel activation precedes and triggers the
expression of myogenin and MEF2, two key transcription factors of the myogenic
differentiation program. Kir2.1 channels are upregulated within the first 6
hours of the differentiation process, i.e. several hours before myogenin and
MEF2 expression (Konig et al.,
2004). To our knowledge, Kir2.1 activation is, so far, the
earliest detectable event during human myoblast differentiation.
In the present study, we evaluated on primary cultures of human myoblasts various potential molecular mechanisms that could control Kir2.1 channel expression and/or activity at the onset of the differentiation process. We tested whether Kir2.1 channels were regulated at the level of protein synthesis, vesicular trafficking (from the endoplasmic reticulum via the Golgi apparatus to the plasma membrane), or whether the activity of the channel at the membrane was modulated.
Several types of ionic channels are known to be expressed or repressed
during developmental programs, and this is also the case of Kir2.1 channels.
For example, several domains in the chicken Kir2.1 promoter regulate the
tissue-specific expression of this channel
(Mutai et al., 2004). It has
also been shown that electrical activity is required for the stability of
Kir2.1 mRNA into skeletal muscle cells
(Shin et al., 1997).
Furthermore, growth factors can regulate the transcription rate of several
muscle channels (for example T-type Ca2+ channel)
(Avila et al., 2006).
With respect to trafficking, Kir2.1 protein contains ER-export motifs that
control the targeting of channels to the plasma membrane. These motifs are
likely to act as recognition signals for its incorporation into COP-II coated
transport vesicles. They were described to be necessary and sufficient for
channel export without need for additional interacting partners
(Ma et al., 2001;
Stockklausner et al., 2001)
(but see Grishin et al.,
2006). Signaling pathways like the Ras-MAPK pathway also appear to
act on Kir2.1 channels trafficking
(Giovannardi et al., 2002). In
addition, binding sites for anchoring proteins such as the filamin A
(Sampson et al., 2003),
PSD93
(Leyland and Dart,
2004) or SAP97 (Leonoudakis et
al., 2004) are thought to stabilize Kir2.1 channels at the plasma
membrane.
Alternatively, the gating properties of ionic channels at the plasma
membrane can be modulated by various intra- and extracellular signaling
pathways. Members of the inward rectifying K+ channel family
possess multiple PIP2-binding sites that are known to be key
regulators of their gating properties
(Rohacs et al., 2003;
Soom et al., 2001).
Furthermore, Kir2.1 can be modulated by PKA
(Wischmeyer and Karschin,
1996), PKC (Fakler et al.,
1994; Jones, 2003)
and receptor-activated tyrosine kinases
(Hoger et al., 2002;
Ruppersberg, 2000;
Ruppersberg and Fakler, 1996;
Wischmeyer et al., 1998).
Recently, it has been suggested that kinases and phosphatases can be
intimately associated to channels in a single regulatory protein complex that
modulates channel activity (Levitan,
2006).
Using whole-cell patch-clamp recording, we found that Kir2.1 channels activated at the onset of the differentiation process are already present at the plasma membrane during proliferation, but that they are silent. Their activity is induced by dephosphorylation of Kir2.1 tyrosine 242.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Cell cultures
Clonal cultures of human myoblasts were prepared from single satellite
cells and expanded as described by Liu et al.
(Liu et al., 1998).
Electrophysiological recordings
Kir2.1 currents were measured in the whole-cell configuration of the
patch-clamp technique as in Konig et al.
(Konig et al., 2004). During
intracellular application of bpV(Phen) through the patch pipette, we decreased
the amount of BAPTA in the intracellular solution to 0.2 mM because vanadates
are sensitive to Ca2+ chelators and Ca2+ concentration
(Huyer et al., 1997). In these
experiments, CaCl2 was added to reach a final concentration of 200
nM, and KCl was increased to maintain osmolarity.
Metabolic labeling
Myoblasts were kept in methionine-free medium for 15 minutes with or
without cycloheximide (3 µg/ml). [35S]-methionine (12.5
µCi/well containing 105 myoblasts) was then added for 30
minutes. Cells were lysed in triton buffer (PBS, Triton X-100 0.5%, 5 mM EDTA)
and proteins were precipitated by TCA before to be mixed with liquid
scintillation cocktail (Ready Value). Radioactivity was measured on
scintillation counter (Beckman Coulter).
DNA construct and transfection
Mutations of the human pcEGFP-Kir2.1 tyrosines 242, 336 and 366 into
phenylalanine residues were carried out with the QuickChange Kit (Stratagene).
Mutations were verified by sequencing. Transfections were performed as
described by Espinos et al. (Espinos et
al., 2001). Proliferating myoblasts were electroporated with
pcEGFP-Kir2.1, with the mutants pcEGFP-Kir2.1Y242F,
pcEGFP-Kir2.1Y336F or pcEGFP-Kir2.1Y366F, or, for
control experiments, with pcEGFP, and maintained 24 hours in growth medium
before electrophysiological recording or other treatments.
Membrane enrichment
Cultured myoblasts were lysed with a hypotonic buffer (20 mM Tris-HCl, pH
7.5, 10 mM NaCl, 1% NP-40) with protease inhibitors and 1 mM
Na3VO4. Nuclei were removed and cell extracts were then
ultracentrifuged for 1 hour at 100,000 g at 4°C. Crude
membranes pellets were resuspended in hypotonic buffer and used for
immunoprecipitation. Protein content was calculated using a Bradford assay
(BioRad).
Immunoprecipitation
Crude membrane proteins (10 mg) from non-transfected cells or total lysates
(100 µg) from transfected cells were precleared on proteinA-sepharose
(Sigma) before incubation with 3.6 µg rabbit anti-Kir2.1 for 2 hours at
4°C. ProteinA-sepharose beads (50 µl) were used to immunoprecipitate
Kir2.1 proteins (overnight at 4°C). The beads were pelleted by
centrifugation (10,000 g for 1 minute), and
SDS-βmercaptoethanol sample buffer was used to separate
immunoprecipitated proteins from the beads (3 minutes at 100°C).
Biotinylation of cell surface proteins
Myoblasts were incubated for 20 minutes with 0.25 mg/ml NHS-Biotin diluted
in PBS at 4°C. NHS-Biotin was then quenched for 15 minutes with 40 mM
NH4Cl/PBS at 4°C. For the separation of biotinylated proteins,
the same amount of proteins from the different lysates was incubated for 1
hour at 4°C with 50 µl of streptavidin magnetic beads (Roche).
Non-biotinylated proteins were separated from the beads with a magnetic
particle separator. The beads were then washed three times with hypotonic
buffer. Biotinylated proteins were recovered in 50 µl of
SDS-βmercaptoethanol sample buffer. After 3 minutes at 100°C, beads
were removed with the magnetic separator.
Western blotting
Aliquots were analyzed by western blot as in Konig et al.
(Konig et al., 2004).
Membranes were incubated with rabbit polyclonal anti-Kir2.1 (1:200), mouse
monoclonal anti-phosphotyrosine (clone PT-66, 1:3000) or chicken polyclonal
anti-Kir2.1 antibodies (1:250). Primary antibodies were then exposed to
horseradish peroxidase-conjugated goat anti-mouse, goat anti-rabbit or donkey
anti-chicken antibodies (1:6000).
TIRF measurement
Tracking of membrane-inserted Kir2.1-GFP channels was obtained from total
internal reflection fluorescence (TIRF) images as in Marthinet et al.
(Marthinet et al., 2005).
Image analysis was performed using MetaMorph software.
Statistics
Results are expressed as the mean±s.e.m. Statistical analyses were
performed using Student's t-test (asterisk in figures indicates
P<0.05).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Fig. 1A illustrates a Kir2.1
current elicited at various potentials and its inhibition by 500 µM
BaCl2. The current-to-voltage relationships show the typical inward
rectification of the Kir2.1 channels (EK=-84 mV in our recording
conditions). In order to determine whether Kir2.1 channel activation at the
beginning of the differentiation process was due to new channel synthesis, we
inhibited de novo protein synthesis with cycloheximide and evaluated the
Kir2.1 channel activity after 6 hours of differentiation
(Fig. 1B). Efficiency of the
drug was established by measuring [35S]-methionine incorporation.
Application of 3 µg/ml cycloheximide for 3 hours blocked 81±6% of
total new protein synthesis. Kir2.1 channel activity was measured in myoblasts
maintained in differentiation medium supplemented with 3 µg/ml
cycloheximide for 6 hours and compared with the Kir2.1 activity measured in
myoblasts kept in control differentiating conditions for the same time. The
fraction of myoblasts maintained in cycloheximide and expressing a Kir2.1
current was not statistically different from that of myoblasts maintained in
control conditions (control=53±9%, n=28,
cycloheximide=34±6%, n=46, P=0.117). The Kir2.1
current density of myoblasts kept 6 hours with cycloheximide was, however,
smaller than that of myoblasts in control conditions (control=-1.1±0.3
pA/pF, n=28, cycloheximide=-0.4±0.1 pA/pF, n=46,
P=0.002, but see below). In parallel experiments, we assessed Kir2.1 channel activity in the presence of 10 µg/ml Brefeldin A, an inhibitor of endoplasmic reticulum (ER) to Golgi transport (Fig. 1C). This was done to evaluate whether the Kir2.1 channel activation during the 6 first hours of differentiation could be due to a translocation of channels from the ER to the plasma membrane. No statistical difference was observed in the presence of the drug when compared with control. The fraction of myoblasts presenting Kir2.1 current (control=42±6%, n=19, Brefeldin A=40±7%, n=46, P=0.862), as well as the Kir2.1 current density, was similar in both conditions (control=-0.7±0.2 pA/pF, n=19, Brefeldin A=-0.5±0.1 pA/pF, n=46, P=0.558). To verify that Brefeldin A at 10 µg/ml was efficiently inhibiting the ER-to-Golgi transport, myoblasts were transfected with the fusion proteins Kir2.1-GFP, and newly synthesized GFP-channels visualized by confocal microscopy. Unlike what was observed in the control conditions, in the presence of the Brefeldin A most fluorescence was located around the nucleus, demonstrating the efficiency of the drug at blocking the transport from the ER to the plasma membrane.
These data strongly suggest that Kir2.1 channel activation at the onset of differentiation is not due to protein synthesis or trafficking regulations, the two sets of experiments complementing and strengthening each other. Indeed, if a new synthesis of Kir2.1 channels would have taken place during the first 6 hours of differentiation, we should have observed a diminution of the Kir2.1 current after treatment with Brefeldin A. As we did not detect any reduction of the current in the presence of Brefeldin A, we conclude that Kir2.1 activation is not due to a new synthesis followed by transport to the plasma membrane. The diminution of Kir2.1 current density observed in myoblasts kept 6 hours with cycloheximide could be due to either an effect of the drug on channel activity or to an intrinsic instability of the required phosphatase (see below), but not on the inhibition of Kir2.1 protein synthesis.
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). To
investigate whether such a mechanism could be involved during myoblast
differentiation, we tested Kir2.1 current activation in the presence of either
bpV(Phen), a tyrosine phosphatase inhibitor, or genistein, a tyrosine kinase
inhibitor. As shown in Fig. 2A,
the proportion of myoblasts expressing a Kir2.1 current is strongly reduced in
the presence of the tyrosine phosphatase inhibitor bpV(Phen). A Kir2.1 current
could be recorded in only 10% (2 out of 19) of the myoblasts placed for 6
hours in differentiation medium supplemented with 10 µM bpV(Phen). By
contrast, in the control differentiation conditions, 49% of the tested
myoblasts had Kir2.1 current (23 out of 47). We should point out that, in the
presence of bpV(Phen), the percent of myoblasts with Kir2.1 current is similar
to that observed during normal proliferation
(Konig et al., 2004). The
current density of bpV(Phen)-treated myoblasts was reduced to one tenth of
that of non-treated myoblasts (P=0.005,
Fig. 2A, inset).
We then added genistein, a tyrosine kinase inhibitor, to the
differentiation medium; application of 10 µM genistein during 4 hours
drastically increased the number of Kir2.1-positive cells. It is important to
mention that Kir2.1 channels are not yet activated after 4 hours in
differentiation conditions (Konig et al.,
2004). Indeed, whereas 45% of the myoblasts (14 out of 31)
presented a Kir2.1 current in the presence of the drug, only 14% of the
myoblasts (3 out of 22) had measurable Kir2.1 current in the control
conditions (Fig. 2B).
Consistently, the current density of genistein-treated myoblasts was fourfold
greater than non-treated myoblasts (P=0.036,
Fig. 2B, inset). From these
observations, we conclude that bpV(Phen) slows down Kir2.1 activation, whereas
genistein accelerates it.
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), we examined whether the inhibition of tyrosine kinases and
phosphatases also affects the differentiation and fusion process. Experiments
were carried out after 24 hours in differentiating conditions, as we wanted to
evaluate the beginning of the fusion process.
Fig. 2C shows that application
of bpV(Phen) slowed down myoblast fusion (down to 2% of fusion,
P=0.02), whereas genistein accelerated it (up to 28% of fusion,
P<0.001), in comparison to the control conditions (5% of fusion).
The early myotubes (24 hours) that are observed after genistein treatment do
not differ morphologically from the early myotubes that appear at a later time
(28 hours) in control cultures, when the differentiation is more advanced
(data not shown). The immunoblot of Fig.
2C confirms that 10 µM bpV(Phen) enhanced the overall myoblast
protein phosphorylation level and that 10 µM genistein reduced it. Altogether, these results show that activation of Kir2.1 channels and the fusion process are induced by a reduction of tyrosine kinase activity and blocked by an inhibition of tyrosine phosphatase activity.
Kir2.1 current activity is reduced by a tyrosine phosphatase inhibitor
The next step was to determine whether the amplitude of the Kir2.1 current
could be modulated by genistein or bpV(Phen) during recording. For that
purpose, either bpV(Phen) or genistein was included in the intracellular
pipette solution during whole-cell patch-clamp recordings.
Fig. 3A (top) illustrates the
stability of the Kir2.1 current under control conditions during a 25 minutes
recording (left traces are after 1 minute recording and right traces after 25
minutes). When 100 µM bpV(Phen) was added to the pipette solution, Kir2.1
current was reduced by 50% of its initial value after 10 minutes recording,
and by 90% after 25 minutes (Fig.
3A, middle traces and Fig.
3B). Finally, co-application of 100 µM bpV(Phen) and 100 µM
genistein through the patch pipette (to slow-down a tyrosine
re-phosphorylation of the Kir2.1 channels) halved the bpV(Phen)-induced
inactivation of the Kir2.1 current (Fig.
3A, bottom traces; Fig.
3B).
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Kir2.1 channels are inhibited by a phosphorylation of tyrosine 242
The modulation of Kir2.1 currents by tyrosine phosphatase and kinase
activities could be the consequence of the phosphorylation of one of the
tyrosines of the Kir2.1 channels itself or it could be linked to a more
complex mechanism involving phosphorylation of co-regulators. To evaluate the
first possibility, we immunoprecipitated Kir2.1-GFP channels with an
anti-Kir2.1 antibody and revealed the tyrosine phosphorylation with an
anti-phosphotyrosine antibody (PT-66). Fig.
4A shows that Kir2.1-GFP channels were tyrosine-phosphorylated,
and that the channel phosphorylation were halved when tyrosine kinases were
blocked by genistein and enhanced fivefold when tyrosine phosphatases were
inhibited by bpV(Phen).
We then replaced the tyrosine 242 of the Kir2.1-GFP channel by a
phenylalanine (Kir2.1-GFPY242F) and tested the effect of bpV(Phen)
on the channel activity. This point mutation was chosen as the tyrosine 242
has been shown to regulate the activity of Kir2.1 channels through its
phosphorylation (Wischmeyer et al.,
1998). We verified that the Kir2.1-GFP and the
Kir2.1-GFPY242F currents behaved electrophysiologically as the
native Kir2.1 current, and that Kir2.1-GFP responded in the same way to
bpV(Phen) treatment. Myoblasts transfected with Kir2.1-GFP and
Kir2.1-GFPY242F displayed typical Kir2.1 currents, except that the
amplitude of the currents was much larger than the endogenous Kir2.1 current
(Kir2.1-GFP=-189±16 pA/pF, n=29;
Kir2.1-GFPY242F=-162±16 pA/pF, n=29).
Fig. 4B shows that, as the
endogenous Kir2.1 current, Kir2.1-GFP current was reduced by at least 90%
after 25 minutes when 100 µM bpV(Phen) was added to the intra-pipette
solution. However, unlike the Kir2.1 channels, the mutant
Kir2.1-GFPY242F did not inactivate in the presence of 100 µM of
bpV(Phen). After 25 minutes bpV(Phen) treatment, the
Kir2.1-GFPY242F current was only slightly decreased (22±8%,
n=3), a reduction that is similar to that observed for Kir2.1-GFP
currents recorded for 25 minutes without bpV(Phen) (17±6%,
n=5). Thus, Kir2.1 channels lacking the tyrosine 242 phosphorylation
site are unaffected by the tyrosine phosphatase inhibitor bpV(Phen). However,
an experiment similar to that illustrated in
Fig. 4A revealed that the
Kir2.1-GFPY242F channel was still tyrosine phosphorylated,
suggesting that other tyrosines were available for phosphorylation (data not
shown). The Kir2.1 primary sequence contains five to seven tyrosine
phosphorylation consensus sites. Therefore, we wondered whether the
phosphorylation of one or several other tyrosines would have an effect on
Kir2.1 activity. We decided to replace two tyrosine residues, Y336 and Y366,
by phenylalanine residues. These tyrosine are the most susceptible to
phosphorylation, as predicted by the NetPhos programme
(http://www.cbs.dtu.dk/services/NetPhos/);
Y242, Y336 and Y366 have phosphorylation scores of 0.976, 0.917 and 0.910,
respectively. Using the Prosite program
(http://www.expasy.org/tools/scanprosite/),
only Y242 and Y366 are potential tyrosine kinase phosphorylation sites. We
also verified that these tyrosines are at the surface of the protein, using
the available structure of the Kir2.1 cytoplasmic part
(Pegan et al., 2005). Kir2.1
current were measured in myoblasts expressing the wild-type Kir2.1-GFP or the
mutated channels, Kir2.1-GFPY242F, Kir2.1-GFPY336F and
Kir2.1-GFPY366F, 1 hour after the addition of 200 µM bpV(Phen)
to the culture medium. Fig. 4C
shows that only the Kir2.1-GFPY242F current was resistant to
bpV(Phen) (P=0.3). By contrast, the wild-type Kir2.1-GFP and the two
mutants, Y336F and Y366F, were strongly inhibited by the bpV(Phen) treatment
(P<0.001).
From these experiments, we conclude that a tyrosine phosphatase inhibition inactivates Kir2.1-GFP current via the phosphorylation of the tyrosine 242 residue of the Kir2.1-GFP channels.
Kir2.1-GFP channels are not internalized during bpV(Phen)-treatment
Phosphorylation of the tyrosine 242 of Kir2.1-GFP channels reduces the
whole-cell Kir2.1-GFP current. This could be the consequence either of a
change in gating properties of the Kir2.1 channel itself (reduction of the
probability of opening) (Wischmeyer et
al., 1998) or of an internalization of the Kir2.1 channels
(Tong et al., 2001). We
evaluated a possible internalization of the channels by bpV(Phen) using the
technique of cell-surface protein biotinylation at 4°C, followed by a
separation of the biotinylated proteins from the non-biotinylated with
streptavidin magnetic beads. The biotinylated fraction of Kir2.1-GFP channels
was evaluated before and after bpV(Phen) treatment (10 µM for 6 hours since
this protocol massively reduced Kir2.1 current;
Fig. 2A, inset).
Fig. 5A shows that the fraction
of Kir2.1-GFP at the plasma membrane was not significantly different after the
6 hours treatment by bpV(Phen) (P=0.91, n=4). Controls were
carried out with non-biotinylated myoblasts and with GFP-transfected
myoblasts.
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Altogether, these results strongly suggest that Kir2.1-GFP current inactivation is not due to an endocytosis of the channels but that it is linked to a direct tyrosine phosphorylation of the Kir2.1 channels.
Differentiation of human myoblasts is linked to a tyrosine dephosphorylation of endogenous Kir2.1 channels
A key experiment of this study was to evaluate endogenous Kir2.1 channels
tyrosine phosphorylation, and also to assess whether this tyrosine
phosphorylation was modulated during differentiation. This experiment was
difficult to perform, as there are only a few hundreds Kir2.1 channels at the
plasma membrane of each myoblasts. Therefore, 300x106
proliferating myoblasts and the same number of myoblast kept for 6 hours in
differentiation medium were harvested. Total lysates were ultracentrifuged to
enrich the membrane fraction. Kir2.1 channels of the enriched membrane
fraction of the two populations were immunoprecipitated using a rabbit IgG
anti-Kir2.1 antibody and revealed on SDS-PAGE with a chicken IgY anti-Kir2.1
antibody. We employed a chicken IgY antibody because the heavy-chain of the
rabbit IgG has the same molecular weight as Kir2.1 channels.
Fig. 6A (upper lane) shows that
endogenous Kir2.1 channels were detectable, and equally present in
proliferation and differentiation conditions. We then reblotted the same
membrane with an antibody against phosphotyrosine residue, which showed that
endogenous Kir2.1 channels of proliferating myoblasts were
tyrosine-phosphorylated, and that this tyrosine phosphorylation was reduced by
50% after 6 hours in differentiation condition
(Fig. 6B).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Konig et al., 2006;
Konig et al., 2004). To our
knowledge, the Kir2.1 tyrosine 242 dephosphorylation is thus the earliest
molecular mechanism described yet in the cascade of events that leads to the
irreversible induction of myoblast differentiation and fusion.
We have previously shown that Kir2.1 mRNA was present in proliferating
myoblasts (Fischer-Lougheed et al.,
2001). We could exclude in the present work that the induction of
Kir2.1 channel activity is due to new channel synthesis or transport of Kir2.1
channels from the endoplasmic reticulum to the plasma membrane. We propose
that, in human proliferating myoblasts, Kir2.1 channels are kept silent at the
plasma membrane by phosphorylation of tyrosine 242, and that these channels
are ready to turn on the differentiation process in response to extracellular
stimuli that induce their dephosphorylation.
Silent Kir2.1 channels are already present at the plasma membrane of proliferating myoblasts
Our results show that upregulation of Kir2.1 channels is not the
consequence of gene expression or trafficking regulation. Neither
cycloheximide nor Brefeldin A had major effects on the activation of Kir2.1
channels during the first 6 hours of differentiation. In addition,
biotinylation and TIRF experiments showed that Kir2.1-GFP channels are still
present at the plasma membrane even after the complete inhibition of Kir2.1
current by bpV(Phen). Together, these observations suggest that Kir2.1
channels are localized at the cell surface of proliferating myoblasts but in a
silent state.
In agreement with this hypothesis, a recent study reported that the active
Kir2.1 channels could represent only a small fraction of the total channels
present at the plasma membrane. Using the FRAC technique (function recovery
after chemobleaching), Sun et al. (Sun et
al., 2004) showed that many `Kir2.1 sleeping molecules' were
present on plasma membrane of HEK293 cells stably overexpressing Kir2.1
channels, and concluded that only a small fraction of channels at the plasma
membrane contributed to the overall channel activity.
Myoblast differentiation is modulated by a tyrosine phosphorylation
Kir2.1 channel increased activity and the fusion process are induced by a
tyrosine kinase inhibitor, genistein, and reduced by a tyrosine phosphatase
inhibitor, bpV(Phen). The interpretation of these pharmacological studies is,
however, complicated by the fact that such inhibitors will affect all
intracellular pathways in which a tyrosine phosphorylation is involved. For
example, genistein inhibits cell proliferation in prostate and breast cancer
cells (Kousidou et al., 2006;
Ouchi et al., 2005). We did
not specifically look at the effect of genistein or bpV(Phen) on human
myoblast proliferation as the drugs were added to cells only under
differentiation conditions. Indeed, our goal was to analyze the early effect
of tyrosine kinase and phosphatase inhibitors on Kir2.1 channel activity and
on the induction of the differentiation process. Although genistein and
bpV(Phen) probably affect many proteins of human myoblasts, these inhibitors
directly target Kir2.1 channels, and modulate the differentiation and fusion
process in a way closely related to the level of Kir2.1 channel
phosphorylation and activity. From these results, we conclude (1) that during
proliferation, a tyrosine kinase activity phosphorylates Kir2.1 channels and
blocks the entry of myoblasts in the differentiation process and (2) that,
although the kinase is still active in the early phase of differentiation, an
increase in tyrosine phosphatase activity targeting Kir2.1 channels is
required to trigger differentiation.
Kir2.1 current is directly modulated by the tyrosine 242 phosphorylation
Evidences for a possible regulation of ionic channels by a direct tyrosine
phosphorylation and dephosphorylation has substantially increased over the
last years (Davis et al.,
2001). Concerning Kir2.1 channels, Wischmeyer et al.
(Wischmeyer et al., 1998)
showed that Kir2.1 channels overexpressed in HEK293 cells were suppressed by
perorthovanadate, and that this inhibition was reversed by genistein. Our
observations in primary cultures of human myoblasts are in good agreement with
these results, the only difference is that the Kir2.1 inhibition by bpV(Phen)
in human myoblasts requires a longer application of the drug to be completed.
Kir2.1 current is inhibited by bpV(Phen) within 10 minutes in HEK293 cells
overexpressing Kir2.1 channels (we confirmed these results ourselves, data not
shown), whereas it required about 25 minutes in human myoblasts. This
difference could be explained either by a lower level of tyrosine kinase
activity in human myoblasts or by a localization of the enzyme that makes it
less accessible to the drug.
The effect of genistein on Kir2.1 current is less striking than that of
bpV(Phen). Application of genistein alone did not result in Kir2.1 currents
activation in proliferating myoblasts. A possible explanation is that the
tyrosine phosphatase that dephosphorylates Kir2.1 channels is lacking or not
activated in proliferating myoblasts. However, co-application of genistein
with bpV(Phen) in differentiated myoblasts expressing Kir2.1 channels reduces
the bpV(Phen)-induced Kir2.1 inhibition. Recently, it has been shown that a
genistein pretreatment blocks the EGF-induced inhibition of Kir2.1 channels in
urothelial cells (Sun et al.,
2007). This observation supports the hypothesis that Kir2.1
channels activity can be directly modulated by a tyrosine phosphorylation.
Finally, we showed that bpV(Phen) did not inactivate the current through
Kir2.1 channels when these channels were mutated on the tyrosine 242. The
mutant mimics a dephosphorylated channel that is constitutively active. This
experiment strongly suggests that the modulation of Kir2.1 channel activity
occurs via the phosphorylation and dephosphorylation of its tyrosine 242.
Differentiation is triggered by the dephosphorylation of endogenous Kir2.1 channels
A major finding of this work is that endogenous Kir2.1 channels are
tyrosine phosphorylated in proliferating myoblasts, and that phosphorylation
is reduced at the onset of differentiation. We therefore propose that Kir2.1
channels are kept silent during proliferation by a tyrosine phosphorylation
due to activated kinase receptors. Several ionic channels have been described
to be inhibited by such mechanism. For example, when Kv1.3 is co-expressed in
HEK293 cells with EGFR, the channel becomes tyrosine-phosphorylated and the
current inhibited, mimicking the regulation of Kv1.3 by Fas in T lymphocytes
(Bowlby et al., 1997).
Similarly, when Kir2.1 channel is co-expressed with EGFR and NGFR in oocytes,
its activity decreases after EGF and NGF exposure
(Wischmeyer et al., 1998).
Recently, it has been shown in bladder urothelial cells that Kir2.1 channel
activity is strongly inhibited by EGF (Sun
et al., 2007). In our human myoblast cultures, one of the main
differences between proliferating and differentiating conditions is a
reduction of growth factors in the differentiation medium. This difference
could, on its own, explain a reduction of tyrosine kinase activity in
differentiating conditions. However, the efficiency of the Kir2.1 channel
dephosphorylation at the onset of differentiation may also depend on the
induction of a tyrosine phosphatase activity. Indeed, recent literature
underscores the major role of tyrosine phosphatases in signal transduction
pathways (Tiganis and Bennett,
2007). The control of the Kir2.1 channel activation throughout
differentiation is thus likely to be dependent on the fine tuning of both
kinase and phosphatase activities. A possible candidate for this
dephosphorylation event could be SHP-2, a tyrosine phosphatase required for
myogenesis (Kontaridis et al.,
2004). SHP-2 has also been proposed to promote slow skeletal
muscle fiber growth by coordinating signals from the extracellular matrix to
the NFAT pathway (Fornaro et al.,
2006).
In conclusion, we propose that during proliferation, endogenous Kir2.1 channels are maintained silent at the plasma membrane by a tyrosine phosphorylation. Then, during the first hours of differentiation, a fine balance between tyrosine kinase and tyrosine phosphatase activities dephosphorylates Kir2.1 channels at tyrosine 242, and thereby triggers the activation of the channels. These results provide the basis for further investigation into the link between extracellular signaling and the induction of a differentiation process through the modulation of ionic channels.
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ACKNOWLEDGMENTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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5 pA) and the lower histogram the current density of the total population
of myoblasts (including myoblasts with no current, i.e. <5 pA) in control
and in cycloheximide conditions. Both histograms are from the same recordings.
Leak currents were estimated either by linear extrapolation from the currents
recorded at -40 and -60 mV or by addition of 500 µM Ba2+ to the
external bath solution, and subtracted to the total current. Twenty-eight
myoblasts (out of three different clones) with a mean cell capacitances of
32±3 pF were recorded under control conditions, and 46 myoblasts (out
of three different clones) with a mean cell capacitances of 27±2 pF in
the presence of cycloheximide. Asterisk indicates P<0.05 (and in
subsequent figures). [35S]-methionine incorporation was performed
in the absence or presence of cycloheximide 3 µg/ml during 3 hours to
evaluate protein synthesis. (C) Myoblasts were cultured in DM for 6
hours with or without 10 µg/ml Brefeldin A (BFA). Histograms represent the
fraction of myoblasts with Kir2.1 current and the Kir2.1 current density as in
B. Both histograms are from the same recordings. Nineteen myoblasts (out of
four different clones) with a mean cell capacitances of 34±3 pF were
recorded in control conditions, and 46 myoblasts (out of three different
clones) with a mean cell capacitances of 23±2 pF in the presence of
Brefeldin A. Representative pictures (confocal microscopy) of myoblasts
transfected with Kir2.1-GFP and incubated immediately after transfection in
the presence or absence of 10 µg/ml Brefeldin A for 12 hours. In the
presence of Brefeldin A, Kir2.1-GFP channels do not reach the plasma
membrane.
