QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 23 January 2008
doi: 10.1242/dev.011387

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hinard, V. Articles by Bernheim, L. Search for Related Content PubMed PubMed Citation Articles by Hinard, V. Articles by Bernheim, L. Social Bookmarking                         What's this?

Initiation of human myoblast differentiation via dephosphorylation of Kir2.1 K⁺ channels at tyrosine 242

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.

View larger version (29K): [in this window] [in a new window]   Fig. 1. Kir2.1 channels are neither newly synthesized nor transported to the plasma membrane during the first 6 hours of differentiation. (A) Typical Kir2.1 current recorded from one myoblast cultured for 6 hours in differentiation medium (DM). Ba2+ (500 µM), as expected, inhibited the current. Voltage-steps were to -40, -60, -80, -100, -120 and -140 mV from a holding potential at -60 mV. Current-to-voltage relationships are shown in control condition and in the presence of 500 µM Ba2+.(B) Myoblasts were cultured in DM for 6 hours with or without 3 µg/ml cycloheximide (CHX). Kir2.1 current amplitude was assessed during a 300 ms step to -120 mV from a holding potential at -60 mV. The upper histogram represents the fraction of myoblasts expressing Kir2.1 current (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.

View larger version (47K): [in this window] [in a new window]   Fig. 2. Kir2.1 channel activation and myoblast differentiation are linked to a tyrosine dephosphorylation. (A) Myoblasts were induced to differentiate for 6 hours in the absence or presence of 10 µM bpV(Phen). Kir2.1 current was recorded during a 300 ms step to -120 mV from a holding potential at -60 mV. The histogram represents the fraction of myoblasts with Kir2.1 current density of increasing amplitudes (from 0 pA/pF to -6 pA/pF). Leak currents were estimated as in Fig. 1A. Cell capacitances were 33±3 pF (n=47) in DM 6 hours and 20±2 pF (n=19) in DM supplemented with bpV(Phen). (Right) Kir2.1 current density in control conditions and when bpV(Phen) is added. This histogram is an alternative representation of the data on the left. (B) Myoblasts were induced to differentiate for 4 hours in the absence or presence of 10 µM genistein. Kir2.1 current was measured and results presented as in A. Cell capacitances were 24±3 pF (n=22) in DM 4 hours and 24±3 pF (n=31) in DM supplemented with genistein. (Right) Kir2.1 current density in control conditions and when genistein is added. This histogram is an alternative representation of the data on the left. (C) Fusion index of myoblasts kept for 24 hours in DM (control conditions), or in DM supplemented with 10 µM bpV(Phen) or with 10 µM genistein during the first 4 hours. Fusion index represents the fraction of nuclei within myotubes. As genistein is prepared in DMSO, we verified that the final DMSO concentration (0.1%) had no effect on the fusion index (n=6; P=0.59, data not shown). Myoblasts were fixed with ice-cold methanol, and stained with Hematoxylin-Eosin. Two different clones were evaluated. Nuclei were counted in three randomly chosen microscope fields for each condition. One microscope field usually contains between 400 and 500 nuclei. A representative picture in each condition is shown. Arrows indicate clusters of nuclei in myotubes. Scale bars: 50 µm. An anti-phosphotyrosine immunoblot on myoblast protein extract shows that 10 µM genistein and 10 µM bpV(Phen) affect overall tyrosine phosphorylation.

View larger version (12K): [in this window] [in a new window]   Fig. 3. Kir2.1 current is inhibited by a tyrosine phosphatase inhibitor. Kir2.1 currents were recorded by whole-cell patch-clamp technique in myoblasts induced to differentiate for 24 hours with a patch pipette containing or not (control) 100 µM bpV(Phen). (A) Kir2.1 currents recorded from myoblasts cultured for 24 hours in DM, in the absence (top) or in the presence (middle) of 100 µM bpV(Phen), or in the presence of both 100 µM bpV(Phen) and 100 µM genistein (bottom). Left traces represent currents recorded after 1 minute and right traces after 25 minutes. Voltage-steps were to -40, -60, -80, -100, -120 and -140 mV from a holding potential at -60 mV. (B) Mean Kir2.1 current amplitude assessed every 10 seconds during a 300 ms step to -120 mV from a holding potential at -60 mV in the absence (n=3) or in the presence (n=5) of 100 µM bpV(Phen), or in the presence of both 100 µM bpV(Phen) and 100 µM genistein (n=3) in the pipette solution. Currents are normalized to the initial current measured 1 minute after breaking the patch. Even though 10 µM genistein was added to the culture medium 2 hours before performing the recordings with both bpV(Phen) and genistein in the pipette, a complete maintenance of channel activity was not achieved.

View larger version (26K): [in this window] [in a new window]   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).

View larger version (26K): [in this window] [in a new window]   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.

View larger version (25K): [in this window] [in a new window]   Fig. 6. Tyrosine dephosphorylation of endogenous Kir2.1 channels at the onset of differentiation. (A) Myoblasts (300x106) were cultured in growth medium (GM) or in differentiation medium for 6 hours (DM 6h). Both populations of myoblasts were treated for 30 minutes preceding lysis with 10 µM bpV(Phen) to avoid unspecific dephosphorylation. An equal amount of proteins (10 mg) from each population was immunoprecipitated with a rabbit anti-Kir2.1 antibody. Immunoprecipitated proteins were separated on SDS-PAGE, and revealed with chicken anti-Kir2.1 antibody (upper lane) and then reblotted with an anti-phosphotyrosine antibody (PT-66, lower lane). Bands were quantified by Optiquant software. (B) The histogram represents the fraction of endogenous Kir2.1 channels that are tyrosine-phosphorylated normalized to the tyrosine-phosphorylated Kir2.1 channels in proliferating conditions. Results were obtained from three independent experiments.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter