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1-syntrophin: association of TRPC1 with dystrophin complex and the PDZ domain of 1-syntrophin
* Institut de Physiologie et Biologie Cellulaire, CNRS, UMR-6187, University of Poitiers, and
LGCM CHU de Poitiers, Poitiers, France
1Correspondence: Institut de Physiologie et Biologie Cellulaires, CNRS, UMR-6187, University of Poitiers, 86022 Poitiers, France. E-mail: bruno.constantin{at}univ-poitiers.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1-syntrophin in muscle cells. TRPC1 was also associated with 1-syntrophin in dystrophic muscle cells independently of dystrophin. Furthermore, glutathione S-transferase (GST) pull-down assays showed that TRPC1 binds to the 1-syntrophin PDZ domain. Transfected recombinant 1-syntrophin formed a complex with TRPC1 channels and restored normal CCEs in dystrophic muscle cells. We suggest that normal regulation of CCEs in skeletal muscle depends on the association between TRPC1 channels and 1-syntrophin that may anchor the store-operated channels to the dystrophin-associated protein complex (DAPC). The loss of this molecular association could participate in the calcium alterations observed in dystrophic muscle cells. This study provides a new model for the regulation of calcium influx by interaction with the scaffold of the DAPC in muscle cells. —Vandebrouck, A., Sabourin, J., Rivet, J., Balghi, H., Sebille, S., Kitzis, A., Raymond, G., Cognard, C., Bourmeyster, N., Constantin, B. Regulation of capacitative calcium entries by 1-syntrophin: association of TRPC1 with dystrophin complex and the PDZ domain of 1-syntrophin.
Key Words: dystrophic muscle • store-operated calcium entries • TRP channels • scaffolding proteins
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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resulting in the degradation of dystrophic muscle fibers. In muscle cells from mdx mice, the lack of dystrophin is associated with an increase in the calcium concentration underneath the plasma membrane (2)
. This is in accordance with the increase of calcium influx across the plasma membrane of dystrophic muscle cells (3
, 4)
. Calcium mishandling in DMD myotubes depends on muscle contractile activity (5)
, suggesting that specific channels activated during these processes may lead to altered calcium entries. Indeed, abnormal calcium influx involves uncontrolled stretch-activated currents (6
, 7)
, and calcium elevations and muscle damage in mdx fibers were prevented by stretch-activated channel blockers (8)
.
Different studies have revealed that capacitative calcium entries (CCEs) support cumulative calcium entries in skeletal muscle fibers and are necessary for long-term activity of muscles (9
, 10)
. These properties suggest that CCEs could be a good candidate for mediating sustained and cumulative calcium entries during activity of dystrophic muscles. Indeed, the early work of Hopf and collaborators (11)
, suggested first that "leak channels" that are over activated in mdx and DMD muscle cells could be store-operated cationic channels and generate elevated CCEs. Moreover, fibers from dystrophic mdx mice show increased store-operated calcium currents (12)
. The use of antisens also clearly demonstrated that store-operated calcium currents recorded in fibers were dependent on the expression level of TRPC1 and TRPC4 channels (12)
. Our recent work performed on transgenic cultured myotubes also suggested that minidystrophin restored normal properties of CCEs (13)
, supporting the idea that dystrophin influences store-operated channels via a direct or indirect linkage.
These features prompted us to determine if restoration of CCEs and calcium handling by minidystrophin was associated with molecular interaction between store-operated channels and the dystrophin-associated protein complex (DAPC). The DAPC provides a linkage between the actin cytoskeleton and the extracellular matrix (ECM) and also build a scaffold for accumulating various signaling molecules. We hypothesized that TRPC1 forming store-operated channels and stretch-activated channels (14)
could be linked to the DAPC through the syntrophins, which are a family of scaffolding proteins that contain multiple protein interaction domains (15
, 16)
. Syntrophins bind transmembrane channels like sodium channels SkM1 and SkM2 (17)
or the potassium channel Kir4.1 (18)
through a PSD95-disc large-Zonula occludens protein (PDZ) domain and are associated with dystrophin and other proteins of the dystrophin family, including utrophin and dystrobrevin (19
, 20
, 21)
. Since the 1-syntrophin is the predominant syntrophin isoform in skeletal and cardiac muscle, we explored the molecular interaction between 1-syntrophin and TRPC1 and determined if expression of 1-syntrophin could regulate CCE in dystrophic myotubes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1-syntrophin was cloned with two tandem N-terminal myc epitope tags in pCMS-enhanced GFP. This was done by isolating a 1878 pb restriction fragment by digestion of the 1-syntrophin FL in myc-pQBl25-fC1, (a gift from Dr. Stephen Gee, Ottawa, Canada) with NheI and NotI restriction enzymes. This fragment was ligated into pCMS-enhanced GFP (a gift from Dr. Philippe Gailly, Brussels, Belgium) with NheI and NotI. This plasmid was named 1-syntrophin-GFP.
Cell culture and transfection
Experiments were performed on three cell lines: Two subclones of the Sol8 myogenic cell line, one dystrophin deficient SolC1dys– and the other expressing minidystrophin SolD6minidys+; the SolC57 cell line expressing native full-length dystrophin. These cell lines were cultured as described previously elsewhere (Marchand et al., 2004). Cells were seeded on glass coverslips in 35 or 100 mm plastic dishes (Nunc Delta, Nunc, Roskild, Denmark). For primary cultures, mouse satellite cells were isolated from hind-limbs muscles of 3- to 5-wk-old female mice. Muscles were rinsed and washed in a calcium- and magnesium-free medium (PBS medium containing: 140 mM NaCl, 2.7 mM KCl, 1.8 mM KH2PO4, and 10 mM Na2HPO4, 37°C) and then transferred in a solution containing Ham’s F-12 medium (Cambrex, East Rutherford, NJ, USA) with 1.5 mg/ml collagenase I (Sigma, St. Louis, MO, USA), 2 mg/ml protease IX (Sigma), and 2 mM HEPES for dissociation (45 min, 37°C) with continuous stirring. The supernatants were centrifuged (20 min at room temperature, 350 g), and the pellets were resuspended in a growth medium Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal calf serum and 10% heat-inactivated horse serum. The cell suspension was then filtered on a nylon netting (pore size 21 µm) in culture flasks and preplated in 100 mm plastic Petri dishes for 1 h (37°C, 5% CO2, water-saturated air) to remove most of the adhering nonmuscle cells. Then, cells were seeded in 25 cm2 culture flasks and again incubated in same conditions. After 3 days, the cells were detached from the flasks by trypsin solution (trypsin and EDTA in Ca2+- and Mg2+-free HBSS, Invitrogen, Carlsbad, CA, USA) and plated on gelatin-coated glass coverslips in 35 mm plastic dishes. The next day the growth medium was exchanged for a fusion medium: DMEM supplemented with 5% heat-inactivated horse serum. All culture media contained penicillin-G (100 U/ml), streptomycin (100 µg/ml), and L-glutamine (2 mM). Cells were transfected 2 days after the initiation of fusion in 100 mm plastic dishes by using JetPEI cationic polymer transfection reagents (Qbiogene, MP Biomedical, Illkirch, France) according to the manufacturer’s instructions. Three micrograms of purified DNA in 100 µl NaCl were added to 6 µl of JetPEI in 100 µl NaCl. The mixture was incubated for 30 min at room temperature and added to 10 ml of growth medium in each dish during 48 h, and then the growth medium was replaced by differentiation medium.
Antibodies
Antibodies against TRPC1: rabbit polyclonal antibody (pAb; Chemicon International, AbCys s.a., Paris, France and Sigma). Antibodies against 1-syntrophin were rabbit pAb to syntrophin alpha 1 (abcam, Cambridge, UK). Other antibodies used were antic-myc (9E10, mouse monoclonal, Sigma); anti-dystrophin (NCL-DYS2, mouse monoclonal (Novocastra, Newcastle, UK). Secondary antibodies for immunofluorescence: RRX-conjugated Affinity Pure Goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA), Fluoprobes 488- anti-mouse IgG antibodies (FluoProbes, Interchim, France). Secondary antibodies for Western blotting were obtained from Amersham Biosciences Corp. (Les Ulis, France): HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG, or from Santa Cruz Biotechnology (Santa Cruz, CA, USA): HRP-conjugated donkey anti-goat IgG.
Recombinant proteins
Vector containing the cDNA of 1-syntrophin was obtained from Stephen Gee (University of Ottawa, Ottawa, Canada). Recombinant proteins were prepared as GST fusion proteins in Escherichia coli (BL21 strain), purified using glutathione-Sepharose beads (Amersham), eluted with glutathione, and used as GST-fusion proteins. The GST-PDZ domain (aa 75–170 of 1-syntrophin) was obtained and produced by means of the plasmid pGEX-5X-3.
Pull-Down Assays
A total of 107 SolD6minidys+ or SolC1dys– myotubes was washed twice in cold PBS and then lyzed in 1 ml of lysis buffer [50 mM Tris HCl, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 1% N P-40 (w/v), 10% glycerol, 5 mM EGTA, 1 mM PMSF, 20 mM leupeptine, 0.8 mM aprotinine, and 10 mM pepstatine]. Glutathione S-transferase (GST; 30 µg) or fusion protein consisting of GST and 1-syntrophin PDZ domain (30 µg) was incubated with 200 µl of each lysates in 600 µl of lysis buffer at 4°C overnight with constant mixing, followed by washing in PBS, pH 7.2. A fraction of the starting material (input) was boiled in SDS-PAGE loading buffer. Beads were washed twice with ice-cold PBS, pH 7.2, and then once with PBS/1% TX-100. The bound proteins were eluted in an equal volume of loading buffer and boiled for 5 min. The input and the bound proteins were fractionated by a 10% SDS–PAGE, followed by Western blotting. The blot was revealed using anti-TRPC1 antibody (Ab).
Immunoprecipitation
All steps were carried out at 4°C or on ice. A total of 107 SolD6minidys+, SolC1dys– or SolC57dys+ myotubes was washed twice in cold PBS and lyzed in 1 ml of radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.05% v/v Nonidet P-40, 1% v/v DOC, 1% v/v TritonX-100, and 0.1% v/v SDS) supplemented with 1 mM PMSF, 20 mM leupeptine, 0.8 mM aprotinine, and 10 mM pepstatine. Muscle were taken and frozen in liquid nitrogen then liquidizer in RIPA lysis buffer. An aliquot of this starting material (input) was boiled in reducing SDS-PAGE sample buffer. 2 µg of Ab was added to 100 µl of lysates diluted with 300 µl of NET (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 0.05% v/v Nonidet P-40) and incubated at 4°C with mixing overnight. Then, 30 µg of protein A/G Sepharose beads (Amersham) were added and incubated an additional 1 h at 4°C with mixing. The immune complexes were collected by centrifugation and washed 3 x 10 min with lysis buffer. Proteins were eluted from the beads by boiling in SDS-PAGE sample buffer. Samples were then centrifuged at 14,000 g for 1–2 min and subjected to SDS-PAGE and Western blot analysis. The blot was treated for 1 h with 1:5000 horseradish peroxydase conjugated anti-mouse antibodies or 1:5000 horseradish peroxydase conjugated anti-rabbit antibodies (Amersham, Les Ulis, France), and developed with an enhanced chemiluminescence kit (Amersham). The apparent molecular of proteins was estimated according to the position on the blot of prestained protein markers (Bio-Rad, Hemel, Hempstead, UK). Digital images of blots are acquired by scanning (Epson stylus Photo RX45) and processed by Adobe Photoshop software for adjustments of brightness and contrast.
Immunological staining
The cultured cells were fixed in TBS/4% paraformaldehyde and permeabilized with TBS/0.5% TritonX-100. Samples were then incubated for 1 h with primary antibodies in TBS (20 mM Tris base, 154 mM NaCl, 2 mM EGTA, and 2 mM MgCl2, pH 7.5) /1% BSA (Sigma). After being washed in TBS, cells were incubated for 30 min in TBS/1% BSA with secondary antibodies. Samples were mounted using Vectashield mounting medium (Vector, Burlingame, CA, USA).
Cytofluorescence analysis by confocal laser scanning microscopy
The immunolabeled samples were examined by confocal laser scanning microscopy (CLSM) using a Bio-Rad MRC 1024 ES (Bio-Rad) equipped with an argon-krypton gas laser. The Cy3 fluorochrome was excited with the 568 nm yellow line, and the emission was collected via a photomultiplier through a 585 nm long pass filter. Data were acquired using an inverted microscope (Olympus IX70) with a x60 water immersion objective [image processing was performed with the LaserSharp software (version 3.0, Bio-Rad) for adjustments of brightness and contrast].
Measurement of Ca2+ influx using Mn2+ quenching of fura 2 fluorescence
Myotubes, plated on glass coverslips, were briefly rinsed with a standard external solution (1.8 Ca2+solution; 130 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 10 mM HEPES, 5.6 mM D-glucose, pH 7.4, with NaOH) and incubated for 30 min at room temperature and then 15 min at 37°C in the same solution supplemented with 3 µM (final concentration) of fura 2-AM (FluoProbes). After being loaded, cells were washed with Ca2+-free solution (130 mM NaCl, 5.4 mM KCl, 0.1 mM EGTA, 0.8 mM MgCl2, 10 mM HEPES, and 5.6 mM D-glucose, pH 7.4 with NaOH) before measurement of calcium influx. Cells were perfused by means of a homemade gravity microperfusion device. Fura 2 loaded cells were excited at 360 nm with a CAIRN monochromator (Cairn Research Limited, Faversham, UK), and emission fluorescence was monitored at 510 nm using a CCD camera (Photonic Science Limited, Robertsbridge, UK) coupled to an Olympus IX70 inverted microscope (x40 water immersion fluorescence objective). The influx of Mn2+ through cationic channels could be evaluated by the quenching of the fura 2 fluorescence excited at 360 nm, i.e., at the isosbestic point. The variation of fluorescence was recorded with the Imaging Workbench 4.0 (IW 4.0) software (Indec BioSystems, Mountain View, CA, USA). The normalization was performed as described elsewhere (4
, 11)
. The rate of loss of fluorescence intensity at 360 nm was divided by the initial fluorescence intensity in the cell measured before manganese ions were added, to correct for differences in the cell size. The quench rate, of the fluorescence intensity, expressed as percent per minute, was estimated using linear regression analysis. The rate of the influx was estimated from the slope during the first 40 s after Mn2+ (50 µM final) addition. Controls were performed by adding 50 µM Mn2+ without previous stimulation to ensure that no quenching of fura 2 fluorescence was observed. In these conditions, the average rate of quenching was <1%/min. Statistical analysis was performed with Origin 5.0 software (OriginLab, Northampton, MA, USA). The difference between the mean values of measured parameters was determined by the Student’s t test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A) or from normal cultured mouse myotubes (Fig. 1B, C
) both expressing full-length native dystrophin. In normal muscles, when TRPC1 was immunoprecipitated (ip: TRPC1 lane) from lysates, immunoblots probed with anti-dystrophin antibodies revealed a band of the same size that the one obtained after immunoprecipitation of dystrophin (ip: dys lane) or of -syntrophin (ip: -syn lane). Thus, the full-length dystrophin was coimmunoprecipitated with both -syntrophin and TRPC1 (Fig. 1A
). This demonstrated that TRPC1 is linked to the dystrophin-based cytoskeleton in adult fibers. It was also possible to coimmunoprecipitate TRPC1 together with full-length dystrophin from protein lysates extracted from normal mouse myotubes (Fig. 1B, C
). Immunoprecipitation with anti-dystrophin Ab probed with anti-TRPC1 antibodies (Fig. 1B
) and reciprocally immunoprecipitation of TRPC1 probed with anti-dystrophin antibodies (Fig. 1C
) showed that both proteins were part of the same molecular complex isolated from normal cultured myotubes. Since we previously developed Sol8 myotubes (SolD6) expressing recombinant minidystrophin (22)
, the association of TRPC1 with minidystrophin was explored in this SolD6 myotubes. TRPC1 or 1-syntrophin was immunoprecipitated from cell lysates and probed with anti-dystrophin antibodies. Figure 1D
shows that minidystrophin was coimmunoprecipitated together with TRPC1 (ip: TRPC1 lane) or with 1-syntrophin (ip: -syn lane). These data demonstrated that TRPC1 form a stable complex with minidystrophin, which is also linking 1-syntrophin.
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The distribution of TRPC1 in myotubes was determined by immunostaining and confocal microscopy. TRPC1 was localized in the cytoplasm and concentrated at the sarcolemma (Fig. 1E, G
) in which it colocalized with dystrophin in control SolC57 myotubes (Fig. 1E, F
). TRPC1 was also observed at the sarcolemma of minidys+ myotubes (Fig. 1G
) together with minidystrophin (Fig. 1H
) and at the sarcolemma of dystrophin-deficient myotubes (Fig. 1I
). The TRPC1 staining was also observed in the cytoplasm in addition to the sarcolemmal distribution. This could represent some TRPC1 channels present in intracellular compartment of developing myotubes. TRPC1 was shown to be exclusively concentrated at the sarcolemma of mouse adult fibers, and it was not detected in the Triton-insoluble fraction (12)
.
TRPC1 is associated with the 1-syntrophin and the PDZ domain
The link of TRPC1 channels with dystrophin prompted us to explore if like voltage-gated sodium channels (17)
, TRPC1 channels could bind to 1-syntrophin through its PDZ domain. The presence of TRPC1 associated with 1-syntrophin was thus investigated in adult mouse skeletal muscle, and normal muscles were compared with the dystrophic ones. Control C57BL/10 mouse muscle lysates were subjected to immunoprecipitation with anti-1-syntrophin and anti-TRPC1 (Fig. 2
A). Western-blots probed with anti-TRPC1 antibodies revealed that TRPC1 was coimmunoprecipitated with 1-syntrophin (ip -syn lane). To explore the presence of the complex in dystrophic muscles, the experiment was reproduced with lysates from mdx dystrophic mice (Fig. 2A
). TRPC1 could be also coimmunoprecipitated with 1-syntrophin (ip: -syn lane) from mdx muscles. This demonstrates that despite the absence of dystrophin, TRPC1 and 1-syntrophin still form a stable complex, although the amount of 1-syntrophin associated with the sarcolemma is reduced in dystrophic muscle (21
, 23)
. Although the amount of immunoprecipitated TRPC1 (ip: TRPC1 lane) was similar in the two muscle types, the amount of coimmunoprecipitated TRPC1 with anti-1-syntrophin Ab (ip: -syn lane) was much higher in C57BL/10 muscle lysates compared with muscle lysates from dystrophic mdx mice. The decreased amount of TRPC1 associated to 1-syntrophin is likely due to the reduction of 1-syntrophin at the sarcolemma in dystrophic muscle cells (21
, 23)
. This reduction in the association of 1-syntrophin with TRPC1 in mdx muscle could be a key parameter for explaining the up-regulation of store-dependent currents supported by TRPC1-TRPC4 in mdx fibers (12)
.
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Coimmunoprecipitation assays were also performed to determine if TRPC1 can form a stable complex with 1-syntrophin in cultured myotubes expressing minidystrophin (Fig. 2B
). Immunoprecipitation of 1-syntrophin probed with anti-TRPC1 antibodies (ip: -syn lane) showed that TRPC1 was coimmunoprecipitated with 1-syntrophin from protein lysates of myotubes expressing minidystrophin (Fig. 2B
). Reciprocally, immunoblots probed with anti-1-syntrophin antibodies (Fig. 2C
) also showed that 1-syntrophin was coimmunoprecipitated when TRPC1 (ip: TRPC1 lane) or minidystrophin (ip: dys lane) were immunoprecipitated. The restoration of normal calcium handling and CCE previously demonstrated in myotubes expressing recombinant minidystrophin (13
, 22)
is thus correlated with the association of TRPC1 channels to a complex containing both 1-syntrophin and minidystrophin.
The interactions of syntrophins with ionic channels usually involve the PDZ domain of syntrophins. We have thus produced the PDZ domain of 1-syntrophin fused to gluthatione S-transferase (GST-PDZ) as bait. Lysates from dys– and minidys+ myotubes were incubated with the fusion proteins GST-PDZ (pull-down assay), and bound proteins were analyzed by immunoblotting and probing with anti-TRPC1 antibodies (Fig. 2D
). A band around 88 kDa corresponding to TRPC1 was detected by immunoblots in the lysates from SolC1, dys–, and SolD6 minidys+ myotubes (input lane) and also in bound proteins to the GST-PDZ (GST-PDZ lane), which demonstrated that the PDZ domain of 1-syntrophin is involved in the binding of TRPC1.
Recombinant 1-syntrophin formed a complex with TRPC1 and localized to the sarcolemma
To explore the functional regulation of store-dependent calcium entries by 1-syntrophin, further experiments were designed to overexpress 1-syntrophin in cultured myotubes. A plasmid vector was constructed to express both 1-syntrophin-c-Myc and enhanced GFP (EGFP) in the same cells. The EGFP expression was used to recognize transfected cells and the c-Myc tag for distinguishing recombinant 1-syntrophin from the native protein. To determine if recombinant 1-syntrophin-c-myc could interact with TRPC1, dys– and minidys+ Sol8 muscle cells were transiently transfected and lyzed for protein extraction. Immunoprecipitation with the antic-Myc Ab (Fig. 3
A) showed that TRPC1 was coimmunoprecipitated with the recombinant 1-syntrophin (ip: c-myc lane) in the two cells types. Reciprocally, when TRPC1 was immunoprecipitated and Western blots were probed with antic-Myc, recombinant 1-syntrophin was coimmunoprecipitated (Fig. 3B
). These experiments showed that TRPC1 forms a complex with the recombinant syntrophin and that the c-Myc tag did not prevent this interaction. The distribution of the native and recombinant 1-syntrophin in myotubes was explored by immunostaining and confocal microscopy. The double staining for 1-syntrophin (in red) and the dystrophin (green) in minidys+ SolD6 myotubes showed colocalization (Fig. 3C
) of these two proteins along the sarcolemma. Dys– SolC1myotubes also displayed peripheric staining of 1-syntrophin (Fig. 3D
) but more sparsely distributed than in myotubes expressing minidystrophin. When transfected with a plasmid also encoding for EGFP, the 1-syntrophin-c-Myc was clearly concentrated at the sarcolemma, while EGFP expression was observed in the cytoplasm (Fig. 3E
). The sparse and lower expression of 1-syntrophin at the sarcolemma of dystrophin-deficient myotubes was confirmed in mdx myotubes (Fig. 3F
) when compared with primary cultures from normal C57bl/10 mice (Fig. 3G
). Minidystrophin expression improved the sarcolemmal distribution of 1-syntrophin when compared with dys– myotubes. However, the sarcolemmal distribution of 1-syntrophin was always more pronounced in C57bl/10 myotubes in primary culture, which reached more differentiated stages than cell lines.
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Forced expression of 1-syntrophin restored normal capacitative calcium entries in dystrophic myotubes
To activate CCEs, the SR was gradually depleted by repetitive activation of calcium release in the presence of a SERCA inhibitor as described previously (13)
. After the stores were depleted by this procedure, 1.8 mM calcium and 50 µM manganese ions were readmitted in the extracellular space, which induced a capacitative influx of divalent cations and a progressive quench by Mn2+ of fura 2 fluorescence emitted from myotubes. (Fig. 4
A). Store-operated cationic influxes measured as the average rate of fluorescence decrease of fura 2 was 
2 times higher in the dystrophic mdx myotubes in primary culture than in normal C57BL/10 myotubes (Fig. 4A, B
).
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CCEs were also measured after the recombinant 1-syntrophin was transiently transfected in normal and mdx myotubes (Fig. 4B
, white columns). The green fluorescence of myotubes (Fig. 3E
) was used to identify transfected cells with pCMS-enhanced GFP plasmid and thus expressing both EGFP and recombinant 1-syntrophin. No significant changes in the rate of quenching were observed in normal C57Bl/10 myotubes expressing recombinant 1-syntrophin compared with nontransfected ones (untransfected cells: –7.40%/min±0.55, n=31 vs. transfected myotubes: –6.36%/min±0.72, n=12). On the opposite, the rate of capacitative influx in mdx was significantly reduced (43% reduction) when recombinant 1-syntrophin was expressed compared with untransfected myotubes (untransfected cells: –16.88%/min±1.19, n=36 vs. transfected myotubes: –9.61%/min±1.35, n=7).
We previously described (13)
that the quench rate were 2 times reduced in Sol8 myotubes expressing minidystrophin compared with dystrophin-deficient SolC1 myotubes. We thus explored the effect of forced expression of recombinant 1-syntrophin on Mn2+ influx measured in this cellular model. As for control C57Bl/10 myotubes, forced expression of 1-syntrophin did not change the rate of quenching in minidys+ SolD6 myotubes (untransfected cells: –15.12%/min±0.88, n=49 vs. transfected myotubes: –13.63%/min±1.81, n=15). The level of calcium influx observed in myotubes expressing recombinant minidystrophin was comparable with the one measured in SolC57 myotubes expressing native dystrophin (13)
. On the contrary, 1-syntrophin expression in SolC1 dystrophin-deficient myotubes lowered CCE, similarly to what was observed with dystrophic mdx myotubes. Transfected dys– myotubes displayed slope of fura 2 quenching that was significantly reduced compared with nontransfected cells (untransfected cells: –30.62%/min±1.51, n=72 vs. transfected myotubes: –9.91%/min±1.80, n=15).
Although no specific blockers for SOCs and TRPC channels are available, the pharmacological inhibitor SKF-96365 has been already used with success as a blocker of CCEs in cultured myotubes from mice (10)
. We thus addressed the sensitivity of store-operated calcium influx in cultured myotubes. The preincubation of SKF-96365 (40 µM) caused a significant inhibition of manganese influx through SOCs in both dys– and minidys+ myotubes (Fig. 4C
, gray columns). This channel blocker was also efficient on capacitative cations influx recorded on myotubes in primary cultures from mdx and C57Bl10 mice (not shown) as previously shown (10)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 25)
as well as in skeletal muscle fibers (12)
. The possibility of functional interaction between TRPC1 and dystrophin has been raised since TRPC1-dependent store-operated currents were found abnormally active in mdx dystrophin-deficient fibers (12)
and because minidystrophin was able to restore normal CCEs in dystrophin-deficient myotubes (13)
. Indeed, TRPC1 has been shown to associate with caveolin-scaffolding lipid raft domains (26)
and the C-terminus tail contains ankyrin-like sequence and coiled-coil domain (dystrophin homology domain), which could allow TRPC1-cytoskeletal interactions. Here we present evidence for the association of TRPC1 channels with the DAPC, since these channels can be coimmunoprecipitated with dystrophin and with 1-syntrophin. It is possible that TRPC1 interacts directly with dystrophin through their homologous coiled-coil motifs, as for the dystrophin-dystrobrevin complex (27)
. However, we show here that TRPC1 can also be immunoprecipitated from dystrophic mdx muscle and dys– cultured myotubes, which means that at least part of the TRPC1 channels are associated with 1-syntrophin independently of dystrophin. Moreover, TRPC1 could be captured by 1-syntrophin-PDZ domain alone in GST pull-down assays. Since this GST-construct is devoid of the dystrophin-binding domains PH2 and SU, the binding of TRPC1 to 1-syntrophin would not be mediated by dystrophin, but rather involved a direct or indirect binding to the PDZ domain. TRPC1 can homodimerize through a N-terminal coiled-coil motif (28)
, but expression studies have indicated that TRPC1 can form heterotetramers with TRPC3, TRPC4, and/or TRPC5 (29
, 30
, 31)
. The binding of TRPC1 to the 1-syntrophin PDZ domain could thus be mediated by associated TRPC4 and/or TRPC5 proteins sharing a C-terminal PDZ-binding motif (32
, 33)
.
All these data suggest that TRPC1 channels are anchored to the scaffold of DAPC and that this molecular interaction is mediated by 1-syntrophin. The GST pull-down assays indicate that the PDZ domain of 1-syntrophin is involved in this interaction. Further studies will be conducted in the future to determine if a direct interaction occurs between the C-terminal domain of TRPC1 and 1-syntrophin and to find the specific sequences involved in this interaction.
We previously demonstrated that expression of minidystrophin in SolC1 dystrophin-deficient myotubes allows the restoration of normal CCEs (13)
. Since store-operated currents have been shown to be dependent on TRPC1 expression in mouse muscle (12)
, it was thus possible that the effect of minidystrophin on CCEs is related to interactions between minidystrophin and TRPC1. Indeed, the present study shows that expression of recombinant minidystrophin in these cells also leads to the building of a complex containing TRPC1 channels, minidystrophin, and 1-syntrophin. This suggests that the anchoring of TRPC1 channels to the scaffold of 1-syntrophin/minidystrophin complex is involved in the better regulation of CCEs. Furthermore, we show here that when dystrophin is absent CCEs are abnormally elevated and forced expression of recombinant 1-syntrophin at the sarcolemma restores normal levels of CCEs in SolC1 myotubes and in mdx myotubes. The coimmunoprecipitation assays also show that in myotubes transfected with recombinant 1-syntrophin, TRPC1 channels are associated in complex with the recombinant 1-syntrophin beside the absence of dystrophin. This result is compatible with the previous observation that syntrophin and dystrobrevin can localize to the membrane in the absence of the dystrophin C-terminal domain (34)
. This can be explained by the fact that 1-syntrophin can interact with other subsarcolemmal proteins like utrophin and cortical actin (19; 35). Alternatively, the binding to TRPC1 itself could be sufficient to localize syntrophin to the sarcolemma. The ability to restore normal CCEs by expression of 1-syntrophin without restoring dystrophin expression shows that the function of 1-syntrophin on SOCs does not depend on a tight connection to dystrophin. It is thus suggesting that in normal muscle cells, dystrophin could exert its regulating effect on CCE indirectly by maintaining the integrity of the DAPC, which allows normal interaction of subsarcolemmal 1-syntrophin with TRPC1. In mdx dystrophic muscle, the alteration of TRPC1-dependent store-operated currents (12)
is thus likely related to the reduction of subsarcolemmal 1-syntrophin due to destabilization of the DAPC. Indeed, we show here that less TRPC1 could be coimmunoprecipitated from mdx muscle suggesting a reduction of the amount of sarcolemmal TRPC1 associated with 1-syntrophin.
These data provide additional clues that underscore the potential signaling role for syntrophin. The knockout of 1-syntrophin has been shown to display no histological alterations as for mdx fibers (36)
. Although up-regulated ß-syntrophin may compensate for the absence of 1-syntrophin in these fibers, these observations indicate that 1-syntrophin may be involved in functions separate from stabilizing the sarcolemma. A regulation of channels by syntrophin has been already proposed by showing that the 
2-syntrophin, in intestinal smooth muscle, regulates the gating of SNC5A mechanosensitive channels by a PDZ domain-mediated interaction (37)
. The control of cationic channels by PDZ-containing scaffolding proteins might be a widespread function of dystrophin-associated protein complexes for controlling transmembrane ions fluxes. Here we propose that the dystrophin-based cytoskeleton, by the intermediate of PDZ-containing 1-syntrophin, maintains normal TRPC1-dependent store-operated influx during activation of calcium release in muscle cells (Fig. 5
).
|
The TRPC1 channels could be directly controlled by the binding to the PDZ domain of syntrophin or alternatively the DAPC may provide the scaffold for assembling a multiprotein signaling complex that modulates the channel activity. Alternatively, the assembly of TRPC1 channels to syntrophin-dystrophin complex may determine the mechanosensitivity, since TRPC1 have been shown to form stretch-activated channels (14)
. Interestingly, these two types of channels, SOC and SAC, are overactivated in dystrophic cells (6
, 7
, 12)
. Moreover, with the use of patch clamp in the cell-attached configuration (38)
, SOCs and SACs have been shown to display the same unitary conductance (between 7 and 8 pS) and current-voltage relationship and to have a similar sensitivity to pharmacological agents such as Gd(3+), SKF-96365, 2-aminoethoxydiphenyl borate, and GsMTx4 toxin. Stimulation with insulin-like growth factor-1 increased the occurrence of the activity of both channel types. Together, these observations suggest that SOCs and SACs might belong to the same population or share common constituents. Although the cell-attached experiments suggest that the store-dependent cationic channels of muscle can also be modulated by membrane stretch, the possible activation of SACs by strains during repeated rounds of contraction and relaxation remains to be evaluated. Potentially, the differences in the modes of activation could result from heteromultimerization of TRPC1 and specific subunit combinations with other TRPCs may form different populations of channels with different properties. In addition, the elevated production of IP3 by PLC during depolarization of dystrophic muscle cells (39)
suggests that TRPC channels could be further activated by diacylglycerol or PKC. In dystrophic muscles, the disruption of the DAPC seems to remove the modulation of TRPC1-channels by 1-syntrophin, which increases calcium influxes. Moreover, the PDZ domain of -syntrophin has been shown to bind heterotrimeric G protein, which regulates the dihydropyridine receptor Ca2+ channels (40)
. These views are in accordance with the current idea that the DAPC provides the scaffold for anchoring and regulating calcium transporters and adhesion-mediated-signaling molecules. The alteration of this fundamental mechanism has specific consequences on calcium homeostasis that may be crucial for explaining physiopathological processes involved in DMD. Indeed, the study on muscle cells lacking mg29 (10)
has shown that a slow cumulative calcium entry through SOC is crucial for long-term calcium homeostasis and treatment with pharmacological agents blocking SOCs and SACs during fatigue protocol has shown that the calcium inflows are involved in force maintaining during repeated stimulation (38)
.
Future studies will be conducted to explore the mechanisms of regulation of SOC by -syntrophin and the dependence of this regulation on the PDZ domain. It is also of interest to determine if SOC are constituted by TRPC heterotetramultimers, which confers multiple modes of regulation and protein association. The association of TRPC4 with TRPC1 and 1-syntrophin should be particularly explored since the down-regulation of both TRPC1 and TRPC4 proteins by antisens reduces store-operated currents in muscle fibers (12)
.
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ACKNOWLEDGMENTS |
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1-syntrophin cDNA, and for helpful discussion. We thank Dr. Corinne Huchet-Cadioux from University of Nantes, France, for providing us the first batch of mdx and C57Bl/10 mice. We thank Françoise Mazin for technical assistance and Elise Mok for her help in editing the manuscript. The authors declare that they have no competing financial interests. Received for publication July 21, 2006. Accepted for publication September 22, 2006.
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