From the Banting and Best Department of Medical
Research, University of Toronto, Charles H. Best Institute,
Toronto, Ontario, Canada M5G 1L6 and
Faculty of Biology,
University of Konstanz, D-78457 Konstanz, Germany
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ABSTRACT |
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The 31-amino acid proteolipid, sarcolipin (SLN), is associated with the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA1). Constructs of human and rabbit SLN and of rabbit SLN with the FLAG epitope at its N terminus (NF-SLN) or its C terminus (SLN-FC) were coexpressed with SERCA1 in HEK-293 T-cells. Immunohistochemistry was used to demonstrate colocalization of NF-SLN and SERCA1 in the endoplasmic reticulum membrane and to demonstrate the cytosolic orientation of the N terminus of SLN. Coexpression of native rabbit SLN or NF-SLN with SERCA1 decreased the apparent affinity of SERCA1 for Ca2+ but stimulated maximal Ca2+ uptake rates (Vmax). The N terminus of SLN is not well conserved among species, and the addition of an N-terminal FLAG epitope did not alter SLN function. Anti-FLAG antibody reversed both the inhibition of Ca2+ uptake by NF-SLN at low Ca2+ concentrations and the stimulatory effect of NF-SLN on Vmax. Addition of the FLAG epitope to the highly conserved C terminus decreased the apparent affinity of SERCA1 for Ca2+ relative to native SLN and decreased Vmax significantly. Mutations in the C-terminal domain showed that this sequence is critical for SLN function. Mutational analysis of the transmembrane helix, together with the additive regulatory effects of coexpression of both SLN and phospholamban (PLN) with SERCA1, provided evidence for different mechanisms of interaction of SLN and PLN with SERCA molecules. Ca2+ uptake rates in sarcoplasmic reticulum vesicles, isolated from rabbit fast-twitch muscle (tibialis anterior) subjected to chronic low frequency stimulation, were reduced by approximately 40% in 3- and 4-day stimulated muscle, with a marginal increase in apparent affinity of SERCA1 for Ca2+. SERCA1 mRNA and protein levels were unaltered after stimulation. In contrast, SLN mRNA was decreased by 15%, and SLN protein was reduced by 40%. Reduced SLN expression could explain the decrease in SERCA1 activity observed in these muscles and might represent an early functional adaptation to chronic low frequency stimulation.
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INTRODUCTION |
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In recent studies (1) we cloned and characterized the sln gene encoding the integral membrane protein sarcolipin (SLN),1 confirming the sequence of the 31-amino acid proteolipid from rabbit determined by Wawrzynow et al. (2). Structural similarities between sln and the pln gene encoding phospholamban (PLN), as well as the homology between the two protein sequences, led us to propose that the two genes are members of a family (1).
PLN is a major regulator of the kinetics of cardiac contractility (3).
It is expressed at high levels in cardiac and slow-twitch skeletal
muscle, where SERCA2a is the predominant SERCA isoform (4). Inhibitory
interactions between transmembrane -helices of PLN and SERCA2a
result in a decrease in the apparent affinity of SERCA2a for
Ca2+ (5). Inhibitory interactions can be reversed by
elevation of cytosolic Ca2+ or by phosphorylation at
residues Ser16 and Thr17 in the cytosolic N
terminus of PLN (6), making PLN a key component in the inotropic
response of the heart to
-adrenergic agonists.
By contrast, the function of SLN has remained unclear. Sarcolipin was discovered because it copurifies with SERCA1 (7). It was first isolated on the basis of its properties as a proteolipid and shown to have a mobility in SDS-PAGE corresponding to a mass of about 6 kDa. Attempts to reveal a function for the proteolipid were contradictory. When purified proteolipid was reconstituted with preparations of SERCA1 deficient in proteolipid, the ratio of Ca2+ translocation to ATP hydrolysis increased over 5-fold (8). However, SLN abolished Ca2+ accumulation, acting like an ionophore when added to the exterior of reconstituted liposomes. In a subsequent investigation of the role of sarcolipin in coupling processes, however, these results were not confirmed, and no effects on coupling ratios were observed (9). The fact that Ca2+ uptake could be induced in microsomal vesicles of mammalian cells transfected only with SERCA1 cDNA suggested that SLN does not play a primary role in Ca2+ uptake (10). SLN expression is muscle-specific and complementary to PLN expression. SLN is expressed most abundantly in fast-twitch skeletal muscle, to a lower extent in slow-twitch skeletal muscle, and to an even lower extent in cardiac muscle, mimicking the expression pattern of SERCA1 (1). Since several of the 10 transmembrane helices in all SERCA isoforms are highly conserved, the conservation of the single transmembrane helices in SLN and PLN suggests that both may interact in a similar way with transmembrane helices in SERCA molecules. In this study, we have explored the possibility that SLN regulates SERCA1 as PLN regulates SERCA2a. We have demonstrated that SLN has a dual effect on Ca2+ transport activity. Like PLN, it decreases the apparent affinity of SERCA1 for Ca2+, inhibiting Ca2+ uptake at low Ca2+ concentrations. However, unlike PLN, SLN increases Vmax, enhancing SERCA1 activity at high Ca2+ concentrations.
In earlier studies, Pette and colleagues (11-15) showed that Ca2+-ATPase and Ca2+ uptake activities decrease by 30-50%, without an alteration in the amount of Ca2+-ATPase protein, after 3-4 days of chronic low frequency stimulation of rabbit fast-twitch skeletal muscle. After chronic low frequency stimulation for 6 to 7 weeks, fast-twitch, fast-fatigable muscles transform into slow-twitch, fatigue-resistant muscles (11). Enhanced fatigue resistance, absence of twitch potentiation, and prolonged contraction and relaxation times are associated with significant increases in Na+/K+-ATPase, PLN, and SERCA2a concentrations and significant decreases in SERCA1, ryanodine receptor, dihydropyridine receptor, and triadin concentrations (12). Our knowledge that SLN is an activator of SERCA1 led us to investigate whether SLN expression is lowered at an earlier stage. Our analysis of the expression of SERCA1 and SLN and of Ca2+ uptake activity in stimulated and control muscles suggests that the decrease in SLN expression is responsible for at least part of the inactivation of SERCA1 activity in low frequency-stimulated fast-twitch skeletal muscle. The modulation of SERCA1 activity by differential expression of SLN appears to represent a novel mechanism of SERCA1 regulation.
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MATERIALS AND METHODS |
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Construction of cDNAs--
The construct used for SERCA1
expression, containing nucleotides 1 to 3008 of the fast twitch
skeletal muscle Ca2+-ATPase cDNA (16), was described
previously (17). For expression of human SLN, nucleotides 42-719 of
the human SLN cDNA (1) were cloned into the blunt-ended
XbaI site of pMT2 (Hu-SLN). The rabbit SLN cDNA was
restricted at nucleotide 113 with StuI and at position 323 with PstI and ligated into the blunt-ended XbaI site of pMT2 (Ra-SLN). A sequence coding for the FLAG epitope (IBI) was
attached to the N terminus of rabbit SLN (NF-SLN), creating the
N-terminal sequence NH2-MDYKDDDDK-M1, and to
the C terminus (SLN-FC), creating sequence
Y31-MDYKDDDDK-COOH. Attachment was through the polymerase
chain reaction (18) using primers with 5' add-on sequences containing a
restriction endonuclease site and a sequence encoding the FLAG epitope.
The NF-SLN construct was further subjected to site-directed mutagenesis using the Quick Change Mutagenesis kit from Stratagene. After verification of the constructs by sequencing, they were cloned into the
blunt-ended XbaI site of the expression vector pMT2 (19). The rabbit PLN clone used in this study was described previously (17).
Immunostaining-- HEK-293 T-antigen cells, grown to a confluency of 70-90%, were split 1:40 and transferred to glass coverslips. Six hours later, the medium was replaced, and cells were transfected with either SERCA1 cDNA, NF-FLAG cDNA, or both. In cotransfection experiments, cDNA concentrations corresponded to a molar ratio of SLN to SERCA1 of 3:1. This ratio, slightly below the ratio (4:1) at which the maximal regulatory effect of SLN was observed, was chosen to ensure that SLN would not be present in excess over SERCA1, leading to artifacts in studies of colocalization. Immunostaining was performed 48-96 h after transfection. The cells were fixed to the glass coverslips with 4% formaldehyde in PBS for 15 min at 25 °C, washed three times with PBS containing 0.5% Triton X-100, and incubated for 30 min at 25 °C with buffer P (PBS supplemented with 2% milk powder and 0.5% Triton X-100). Incubation with primary antibody in buffer P was for 1 h at 25 °C, using monoclonal antibody A52, which recognizes a cytoplasmic epitope of SERCA1 (20), monoclonal antibody A20, specific for a luminal epitope of SERCA1 (21), or anti-FLAG antibody M2 (IBI). Glass coverslips were washed three times with buffer P, followed by incubation with secondary antibody (fluorescein-conjugated goat anti-mouse IgG, Molecular Probes). After three wash steps with buffer P, the cells were mounted on glass coverslips using Slow Fade Antifade kit (Molecular Probes).
For selective permeabilization of the plasma membrane, Triton X-100 was replaced by digitonin at a concentration of 50 µM or by 0.05% saponin. Streptolysin O (Sigma) was also used to permeabilize the plasma membrane exclusively. After fixation, cells were incubated for 15 min at 37 °C with PBS containing 5 units/ml streptolysin O. Glass coverslips were then washed with PBS supplemented with 2% milk. In colocalization experiments, rabbit polyclonal antibody C4 against SERCA proteins (22) and anti-FLAG antibody M2 were used simultaneously. FITC-conjugated goat anti-mouse IgG (H + L) and Texas Red-conjugated goat anti-rabbit IgG (H + L) (Molecular Probes) were used as secondary antibodies. Samples were analyzed with a confocal microscope (Carl Zeiss), using two lasers for excitation of fluorescein at 488 nm and Texas Red at 568 nm. The emission signals for FITC (525 nm) and Texas Red (615-620 nm) were captured with a band pass at 510-540 nm and at 590 nm. Images were analyzed using Adobe Photoshop software.Expression of cDNAs and Preparation of
Microsomes--
HEK-293 T-cells were cotransfected with SERCA1
cDNA and SLN construct cDNAs according to the calcium phosphate
precipitation method (23). In a typical coexpression experiment, a
molar ratio (SLN cDNA to SERCA1 cDNA) of 5:1 was chosen to
achieve maximal modulation by SLN. A total amount of 15 µg of DNA was
used for transfection of cells in a 10-cm dish, with pMT2 DNA being
added to adjust total DNA concentration. In experiments coexpressing SERCA1, Ra-SLN, and PLN in various ratios, a total DNA concentration of
17 µg was chosen. A ratio of 1:3:5 of SERCA1:PLN:Ra-SLN led to
saturation of the effect of SLN and PLN on KCa.
When PLN was expressed with SERCA1 in the absence of SLN, pMT2 control
DNA was added to adjust total DNA concentrations. HEK-293 microsomes were prepared (10), quick-frozen, and stored at 70 °C for further analysis.
Immunoblotting and ELISA-- To analyze expression of SERCA1, 15 µg of microsomal proteins were solubilized in SDS buffer and separated on 7.5% PAGE. Proteins were transferred electrophoretically to nitrocellulose and incubated with antibody A52 against SERCA1. Expression of NF-SLN was detected by separation of 15 µg of microsomal proteins on Tris/Tricine gels (24) and subsequent transfer to PROTRANTM nitrocellulose with a pore size of 0.05 µm (Schleicher & Schuell). The membranes were incubated with anti-FLAG antibody (IBI), and antibody binding was visualized using the ECL Western detection system (Amersham Pharmacia Biotech).
SERCA1 was quantitated by a sandwich ELISA (21) in which sheep polyclonal antibody R4 against SERCA1 was used for coating; monoclonal antibody A52 against SERCA1 was used as primary antibody; and a goat anti-mouse alkaline phosphatase conjugated antibody (Promega) was used for detection.Ca2+ Transport Assay and Data Analysis-- Ca2+ dependence of Ca2+ uptake was measured as described previously (17). To determine Vmax, Ca2+ uptake was measured at pCa 5.5, and the concentration of SERCA1 was determined by ELISA. In all experiments where Vmax was evaluated, transfection, microsome preparation, ELISA, and transport assays were always performed simultaneously for SERCA1 alone and for SERCA1 in the presence of NF-SLN or additional SLN constructs. Vmax values were calculated relative to SERCA1 alone. The modulatory effects of NF-SLN on SERCA1 Ca2+ uptake activity were confirmed by dissociation of the two proteins with anti-FLAG antibody. Microsomes (1 mg/ml) were incubated with anti-FLAG antibody M2 (0.5 mg/ml) for 30 min at 4 °C prior to uptake. Control microsomes contained equal amounts of heat-inactivated antibody to ensure identical buffer composition.
Data were analyzed by nonlinear regression using the Sigmaplot Scientific Graph System obtained from Jandel Scientific. K0.5 values were calculated using an equation for a general cooperative model for substrate activation. Vmax values were taken directly from the experimental data.Animals and Chronic Low Frequency Stimulation-- Adult male New Zealand White rabbits were subjected to chronic low frequency stimulation (10 Hz, impulse width 0.15 ms, 24 h/day) as described previously (12). Animals were killed after 3 or 4 days of stimulation, and both stimulated and unstimulated contralateral (tibialis anterior) muscles were excised and frozen in liquid nitrogen.
Isolation of Rabbit Sarcoplasmic Reticulum--
Sarcoplasmic
reticulum was isolated from 300 to 500 mg of frozen contralateral
control muscle and stimulated muscle. Frozen muscle was powdered under
liquid nitrogen in a precooled mortar and suspended in 2 ml of buffer A
(10 mM imidazole, pH 7.0, 0.3 M sucrose, 0.5 mM DTT, 40 µM CaCl2, and protease
inhibitors (Boehringer Mannheim, complete, EDTA-free)). The samples
were homogenized by 10 strokes in a Teflon homogenizer and centrifuged
for 10 min at 8,000 × g. The pellet was rehomogenized
in 2 ml of buffer A and centrifuged for 10 min at 8,000 × g, and the combined supernatants were centrifuged for 10 min
at 15,000 × g. The combined supernatant was then
centrifuged for 1 h at 200,000 × g, and the
microsomal pellet was suspended in 1 ml of buffer B (20 mM
Tris-HCl, pH 7.5, 0.6 M KCl, 0.3 M sucrose, 0.5 mM DTT, 40 µM CaCl2, and protease inhibitors) and centrifuged for 1 h at 200,000 × g. The pellet was suspended in buffer C (10 mM
Tris-HCl, pH 7.5, 150 mM KCl, 0.25 M sucrose,
0.5 mM DTT, 20 µM CaCl2, and
protease inhibitors) and washed once more in buffer C, and microsomal
protein was quantitated using bovine serum albumin as standard.
Concentrations were adjusted to 1 mg/ml, and microsomes were
quick-frozen in liquid nitrogen and stored at 70 °C.
Quantitation of SERCA1 and SLN-- SERCA1 was quantitated by ELISA as described above. To evaluate the relative ratio of SLN to SERCA1 in each preparation, 30 µg of microsomal proteins were loaded on a Tris/Tricine gel (24), and the gel was fixed in 5% formaldehyde and 25% ethanol, washed three times for 10 min with water, and stained with Coomassie Blue R250 for 2 h and destained. The wet gels were analyzed by scanning densitometry using NIH Image 1.59 software. In addition, 4 µg of total proteins were separated by SDS-PAGE using 7.5% gels (25), and the band at 110 kDa was analyzed by densitometry. SLN was purified from rabbit fast-twitch skeletal muscle using the butanol extraction method (26). To verify that the 6-kDa protein that was down-regulated in chronic low frequency-stimulated muscle sarcoplasmic reticulum was indeed SLN, proteins separated on Tris/Tricine gels were transferred to polyvinylidene difluoride membranes, and the 6-kDa protein adhering to the membrane was sequenced by Edman degradation by Santosh Kumar and Dr. Ruedi Aebersold, University of Washington.
Reverse Transcriptase-PCR--
Total RNA was isolated from
electrostimulated or control muscle using
guanidinium/isothiocyanate/phenol/chloroform extraction (27). The RNA
concentration was determined by measurement of absorption at 260 nm,
and quality was monitored by Northern blot analysis. Moloney murine
leukemia virus reverse transcriptase and random primers were used to
reverse transcribe 8 µg of total RNA in a 50-µl reaction as
described previously (1). PCR was carried out in a total volume of 30 µl with Taq polymerase and Taq Extender
(Stratagene) according to the manufacturer, with 200 µM
dNTPs, 1 µCi of [-32P]dCTP, 120 ng of the
appropriate 5' and 3' cDNA primers (SERCA1: forward (F), 5'
CAAAGGCGTCTATGAGAAGGTG 3' and reverse (R), 5' TGCAGGTATTCCACGATCTTGG
3'; SLN: F, 5' ACACAGCCTGGGAGATGAGCC 3' and R, 5'
GAATGAAATAAGTGTACTATCG 3';
-actin: F, 5' AATGGCTCCGGCATGTGCAAG 3'
and R, 5' ACCGCAGCTCGTTGTAGAAGG 3'; glyceraldehyde phosphate dehydrogenase (GAPDH): F, 5' TCATTGACCTCCACTACATGG 3' and R, 5' TGGCACTGTTGAAGTCGCAG 3'), and 1 µl of a 15-fold dilution of first strand cDNA. Denaturation was for 1 min at 94 °C, annealing was at 60 °C for 2 min, and extension was at 72 °C for 3 min. SERCA1,
-actin, and GAPDH cDNAs were amplified for 24 cycles and SLN cDNAs for 32 cycles. The number of cycles was chosen to obtain amplifications within the linear exponential range. An aliquot of 20 ng
of previously amplified unlabeled PCR product was added to 15 µl of
each radioactively labeled PCR reaction mixture, and the spiked
products were separated on a 1.5% agarose gel containing ethidium
bromide. Bands were visualized under UV light and excised, and the
radioactivity was measured in a scintillation counter.
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RESULTS |
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Orientation of NF-SLN in the Sarcoplasmic Reticulum Membrane-- To confirm the assumption that the N-terminal sequence of SLN faces the cytosol and the C terminus protrudes into the lumen of the sarcoplasmic reticulum (1), we immunostained cells coexpressing SERCA1 and NF-SLN, an SLN construct with a FLAG epitope attached to its N terminus. Expression of SERCA1 was detected with either monoclonal antibody A52, recognizing a cytosolic epitope of SERCA1 (20), or with antibody A20, recognizing a luminal epitope of SERCA1 (21). Expression of NF-SLN was detected using antibody M2 specific for the FLAG epitope. Primary antibody was visualized using FITC-labeled goat anti-mouse IgG (H + L). About 30% of total cells were stained after complete permeabilization of cells using 0.5% Triton X-100 with either of the three antibodies. The transfection efficiency was 31 ± 3% (Table I). About the same number of fluorescent cells were detected with anti-FLAG antibody after treatment with 50 µM digitonin to permeabilize the plasma membrane selectively, leaving the sarcoplasmic reticulum membrane intact. Under these conditions a comparable signal was obtained with antibody A52 against the cytosolic epitope of SERCA1, whereas antibody A20 was unable to interact with its luminal epitope. Similar results were obtained in experiments where the plasma membrane was permeabilized with 0.05% saponin or when cells were incubated with 5 units/ml streptolysin O for 15 min at 37 °C (Table I). These results strongly support the hypothesis that the N-terminal sequence of SLN faces the cytosol (Fig. 1).
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Colocalization of SERCA1 and NF-SLN-- HEK-293 T-cells coexpressing SERCA1 and NF-SLN were subjected to immunostaining using rabbit polyclonal antibody C4 against SERCA1 (22) and anti-FLAG antibody as primary antibodies and Texas Red-conjugated anti-rabbit antibody or FITC-conjugated anti-mouse antibody as secondary antibodies. Analysis by confocal microscopy showed a similar staining pattern, with intense staining of both proteins in the sarcoplasmic reticulum membrane but not in the plasma membrane (Fig. 2). An overlay of both pictures revealed colocalization of SERCA1 and SLN. These results are in line with previous findings that SLN copurifies with SERCA1 (7).
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Expression of SERCA1 and SLN Constructs in HEK-293 T-cells-- To determine the effects of SLN on SERCA1 Ca2+ transport activity, we coexpressed SERCA1 and SLN constructs in HEK-293 T-cells, prepared sarcoplasmic reticulum microsomes, and determined Ca2+ dependence of Ca2+ uptake. Both human SLN (Hu-SLN) and rabbit SLN (Ra-SLN) decreased the apparent affinity of rabbit SERCA1 for Ca2+ to the same extent, indicating that the differences observed in the N-terminal sequences of Hu-SLN and Ra-SLN are not critical for SLN function. KCa (the negative logarithm of the Ca2+ concentration that gives half-maximal Ca2+ transport activity expressed in pCa units) was shifted from pCa 6.46 to 6.29 when either Hu- or Ra-SLN were cotransfected with SERCA1 cDNAs in a 5:1 molar ratio (Fig. 3 and Table II). To evaluate the effect of SLN constructs on Ca2+ uptake rates, SERCA1 protein was determined by a quantitative sandwich ELISA (21), and specific Ca2+ uptake activity was calculated. Ra-SLN stimulated maximal Ca2+ transport rates by 40% (p < 0.01). Fig. 4 illustrates the dual effect of Ra-SLN on SERCA1 Ca2+ transport. SLN decreases apparent Ca2+ affinity, thereby lowering Ca2+ uptake rates at low Ca2+ concentrations, but, at saturating Ca2+ concentrations, Vmax is stimulated.
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Identification and Quantitation of SLN in Fast-twitch Skeletal Muscle Sarcoplasmic Reticulum-- To achieve optimal resolution of proteins in the low molecular weight range, we separated microsomal proteins on Tris/Tricine gels (24). The band corresponding to SLN migrated slightly faster than the aprotinin standard band at 6.5 kDa, in line with previous observations that SLN migrates at 6 kDa (7, 26). Different concentrations of rabbit microsomal proteins were separated on Tris/Tricine and Laemmli gels, and the bands in Coomassie R250-stained gels corresponding to SLN or SERCA1 were analyzed by densitometry. Comparison of the signals for SLN and SERCA1, taking into account the 33-fold lower molecular weight of SLN, revealed a molar ratio of SLN to SERCA1 of 0.75. In our preparation of washed sarcoplasmic reticulum membranes, we observed only a single band at 110 kDa. However, we did not have independent confirmation of the purity of the band, and any contamination would lead to an overestimation of SERCA1 concentration and to a ratio between SLN and SERCA1 that could be closer to 1:1. The identity of SLN and its purity in the band indicated in Fig. 5 were proven by separation of sarcoplasmic reticulum proteins on Tris/Tricine gels, transfer to polyvinylidene difluoride membranes, and sequencing of the 6-kDa band by Edman degradation. A clean sequence, identifiable as the first 14 amino acids of SLN, was obtained. When SLN was purified according to the butanol extraction method (26), the isolated proteolipid had the same mobility as the 6-kDa band observed in microsomal proteins (Fig. 5).
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Expression of SLN in Chronic Low Frequency-stimulated Fast-twitch Skeletal Muscle Sarcoplasmic Reticulum-- Although neonatal rabbit skeletal muscles express high levels of SERCA2, SERCA1a predominates in adult skeletal muscle (29). This pattern is reversed by chronic low frequency stimulation (11-15). To investigate the potential role of SLN in the diminished SERCA1 activity observed after chronic low frequency stimulation (11-15), we isolated microsomes from 3- or 4-day stimulated and contralateral normal rabbit fast-twitch skeletal muscles (tibialis anterior). The amount of SERCA1 protein did not change after 3 or 4 days of stimulation, as determined by a quantitative sandwich ELISA and scanning densitometry of protein gels, confirming earlier findings (12, 13, 30). Maximal Ca2+ uptake rates (Vmax) at pCa 5.5 in sarcoplasmic reticulum from stimulated muscle were 60 ± 16% of control muscle at 3 days and 57 ± 14% of control at 4 days. The values from 3- and 4-day stimulation, however, were not significantly different (n = 3). These findings confirm earlier reports showing a 30-50% reduction in Ca2+-ATPase activity (11-15, 30). Measurements of Ca2+ dependence of Ca2+ uptake revealed a slightly increased KCa value after stimulation, from pCa 6.53 ± 0.03 to pCa 6.58 ± 0.03 after 3 days and from 6.53 ± 0.05 to pCa 6.58 ± 0.06 after 4 days. The observed increase in KCa was statistically significant at p < 0.05 for 3-day stimulated muscle, whereas the enhanced KCa value for 4-day stimulated muscle represented only a trend (n = 3; p = 0.09).
Preparations of total RNA from contralateral normal and stimulated muscle were used in reverse transcriptase-PCR reactions to assess possible changes in SERCA1 and SLN mRNA expression (n = 3). GAPDH and
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DISCUSSION |
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Role of SLN as a Regulator of SERCA1 Function-- HEK-293 T-cells have proven to be a suitable system in which to study the functional interaction between SLN and SERCA1, since there is no endogenous expression of these two proteins in kidney cells (1, 31). We have used this expression system to determine that SLN is oriented in the endoplasmic reticulum with its N terminus facing the cytoplasm. A FLAG antibody recognized the FLAG epitope inserted into the N terminus of SLN (NF-SLN) under conditions where plasma membranes, but not endoplasmic reticulum membranes, in transfected HEK-293 T-cells were permeabilized with digitonin, saponin, or streptolysin O, which also permitted staining of a known cytoplasmic epitope in SERCA1, but not of a known luminal epitope in SERCA1. By contrast, all epitopes were recognized in Triton X-100-treated cells in which both plasma membranes and endoplasmic reticulum membranes were disrupted.
Immunostaining of HEK-293 T-cells cotransfected with SERCA1 and NF-SLN also revealed very similar expression patterns for both proteins, with high expression in the endoplasmic reticulum membrane system but no expression in the plasma membrane (Fig. 2, A and B). An overlay of both pictures provided evidence for colocalization of SERCA1 and SLN to the endoplasmic reticulum membrane (Fig. 2C). Copurification of SLN and SERCA1, after solubilization of sarcoplasmic reticulum membranes with deoxycholate and subsequent precipitation with ammonium acetate (7, 9), also suggests that SLN and SERCA1 are closely associated. We showed earlier that SLN is highly expressed in fast-twitch skeletal muscle sarcoplasmic reticulum but is expressed at about 10-fold lower concentrations in slow-twitch skeletal muscle sarcoplasmic reticulum, mimicking the expression pattern of SERCA1 (1). By contrast, PLN is highly expressed in heart muscle sarcoplasmic reticulum and in slow-twitch muscle sarcoplasmic reticulum, mimicking the expression pattern of SERCA2a (4). These studies have led us to propose that SLN may act as the counterpart of PLN in fast-twitch skeletal muscle by regulating SERCA1 function. We obtained evidence for this postulate by coexpression of SERCA1 and SLN in HEK-293 T-cells. Under these conditions, we found a decrease in the apparent affinity of SERCA1 for Ca2+ ofRole of SLN in Muscle Disease-- If mutations in SLN, leading to gain of inhibition of SERCA1 activity (eg. mutant Y29A), should occur naturally, removal of Ca2+ from the cytoplasm would be delayed, and muscle relaxation times would be prolonged. Patients with Brody disease, an inherited disorder of skeletal muscle function, suffer from exercise-induced impairment of muscle relaxation, stiffness, and cramps (37-39). In cultured muscle cells from Brody patients, resting Ca2+ concentrations and the increase in intracellular Ca2+ concentration after addition of acetylcholine were normal, but the time required to reach resting intracellular Ca2+ levels after Ca2+ release was increased severalfold (39). Mutations in the ATP2A1 gene, leading to truncation and loss of function of SERCA1 protein, have been associated with the autosomal recessive inheritance of Brody disease (1, 40). In dominantly inherited forms of the disease, about 50% of currently known cases, no mutations were found in ATP2A1 encoding SERCA1 nor in the sln gene encoding SLN in three patients with dominant forms of Brody disease (1).
Role of SLN in Fast- to Slow-twitch Fiber Conversion-- Chronic low frequency stimulation for several weeks has been shown to transform fast-twitch, fast-fatigable muscles into slow-twitch, fatigue-resistant muscles in rabbit (11). As early as 5 min after onset of stimulation, a pronounced reduction in twitch force generation and electromyographic activity occurred, which gradually recovered over 6 days or more of continued stimulation (12, 41). Metabolic perturbations observed during the 1st h of stimulation, such as a decrease in phosphocreatine and ATP content, a depletion of glycogen, and an increase in lactate, were found to recover after 1 day of continued stimulation (42). Despite the recovery of metabolic changes and force output with stimulation periods longer than 6 days, contraction and relaxation times were prolonged, and intracellular Ca2+ concentrations were enhanced (43). The increase in cytosolic Ca2+ can be explained by a drop in SERCA1 activity, which was observed as early as 1 day after onset of stimulation. After 3 days of stimulation, Ca2+ uptake into sarcoplasmic reticulum microsomes was diminished by 30-50% (11-15). SERCA1 protein content did not change, however, during the first 6 days of stimulation (12, 13). After that, fast-twitch fibers transformed into hybrid fibers expressing both SERCA1 and SERCA2a. SERCA1 expression gradually decreased, while SERCA2a levels gradually increased, and, after 42 days of stimulation, reached levels comparable to those found in slow-twitch muscle (44).
In this study we have confirmed that a 40% decrease in Ca2+ uptake activity occurs in sarcoplasmic reticulum vesicles from fast-twitch muscle (tibialis anterior) stimulated for 3 or 4 days, without a decrease in SERCA1 mRNA or SERCA1 protein. We did, however, find a 40% decrease in SLN content after 3 days and a decrease of 15% of SLN mRNA expression in the same tissue preparation. On the basis of our findings from coexpression of SLN and SERCA1 in cell culture, a reduction in SLN expression would be expected to diminish Vmax in SERCA1, explaining, at least in part, the reduced Ca2+ uptake rates in low frequency-stimulated muscle. The drop in SERCA1 Ca2+ transport activity due to reduced SLN expression soon after onset of stimulation could also explain the rise in intracellular Ca2+ concentration. Such a rise in intracellular Ca2+ may be important in initiating other processes in the transformation of fast- to slow-twitch fibers. The decrease in SLN protein expression was more pronounced than the reduction of SLN mRNA, suggesting that SLN expression is regulated at the levels of both transcription and translation. Changes in the state of protein oxidation, especially tyrosine nitration (45), or changes in the expression of other proteins may also contribute to the observed inactivation of SERCA1. In addition to the reduced SLN expression, the 50% reduction in ![]() |
ACKNOWLEDGEMENTS |
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We thank Santosh Kumar and Dr. R. Aebersold, University of Washington, Seattle, for protein sequence determination; Dr. W. J. Rice, Rockefeller University, New York, for helpful advice on the ELISA protocol; Dr. R. J. Kaufman, Genetics Institute, Boston, for the gift of the pMT2 vector; and Stella deLeon for expert technical assistance.
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FOOTNOTES |
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* This work was supported by grants from the Medical Research Council of Canada, the Muscular Dystrophy Association of Canada, the Canadian Genetic Diseases Network of Centers of Excellence (to D. H. M.), and by Grant Pe 62/19-3 from the Deutsche Forschungsgemeinschaft (to D. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Postdoctoral Fellow of the Medical Research Council of Canada.
¶ Postdoctoral Fellow of the Heart and Stroke Foundation of Canada. Present address: EMBL Outstation, c/o ILL, BP 156, F-38042 Grenoble Cedex 9, France.
** To whom correspondence should be addressed: Banting and Best Department of Medical Research, University of Toronto, Charles H. Best Institute, 112 College St., Toronto, Ontario, Canada M5G 1L6. Tel.: 416-978-5008; Fax: 416-978-8528; E-mail: david.maclennan{at}utoronto.ca.
1 The abbreviations used are: SLN, sarcolipin; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PLN, phospholamban; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; FITC, fluorescein isothiocyanate; Hu, human; Ra, rabbit; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; GAPDH, glyceraldehyde phosphate dehydrogenase.
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REFERENCES |
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