Sarcolipin Regulates the Activity of SERCA1, the Fast-twitch Skeletal Muscle Sarcoplasmic Reticulum Ca2+-ATPase*

Alex OdermattDagger §, Stefan BeckerDagger , Vijay K. KhannaDagger , Kazimierz KurzydlowskiDagger , Elmi Leisnerparallel , Dirk Petteparallel , and David H. MacLennanDagger **

From the Dagger  Banting and Best Department of Medical Research, University of Toronto, Charles H. Best Institute, Toronto, Ontario, Canada M5G 1L6 and parallel  Faculty of Biology, University of Konstanz, D-78457 Konstanz, Germany

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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 beta -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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 [alpha -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'; beta -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, beta -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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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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Table I
Cytosolic orientation of the N terminus of NF-SLN
Transfected cells were permeabilized and incubated with antibodies against SERCA1 or the FLAG epitope attached to the N terminus of NF-SLN. Immunostaining of SERCA1 and NF-SLN was carried out as described under "Materials and Methods." Numbers represent the percentage of fluorescent cells relative to total cells from three independent experiments. In a typical experiment, 200-250 cells were counted (n = 3). The transfection efficiency was 31 ± 3%. Data are presented as mean ± S.D.


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Fig. 1.   Circle model of SLN and PLN. Black circles represent identical amino acids, and shaded circles represent amino acids conserved between SLN and PLN. Amino acids are indicated in a single letter code. Domains of SLN and PLN are designated on the left, and numbers indicate their positions in the sequence.

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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Fig. 2.   Colocalization of NF-SLN and SERCA1 coexpressed in HEK-293 T-cells. HEK-293 T-cells were cotransfected with SERCA1 and NF-SLN cDNAs in a molar ratio of 1:3. Immunostaining was performed 72 h post-transfection. The cells were dual labeled with rabbit polyclonal antibody C4 against SERCA1 and mouse anti-FLAG antibody against the FLAG epitope of NF-SLN, in the presence of 0.5% Triton X-100. Subsequently, the cells were incubated with Texas Red-conjugated goat anti-rabbit IgG and FITC-conjugated goat anti-mouse IgG. A, emisson of Texas Red fluorescence from labeled SERCA1 at 615-620 nm. B, emission of FITC fluorescence from labeled NF-SLN at 525 nm. C, an overlay of A and B demonstrating colocalization.

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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Fig. 3.   A, effects of wild-type SLN, mutant SLN, and PLN on the affinity of SERCA1 for Ca2+. For each mutant, the normal amino acid residue is defined on the left; the position in the sequence is indicated by a number, and the newly introduced amino acid residue is defined on the right. Amino acids are indicated in a single letter code. KCa, expressed in pCa units, is the negative logarithm of the Ca2+ concentration at which half-maximal Ca2+ uptake rates were observed. The vertical dashed line on the left represents the KCa value for SERCA1, and the vertical dashed line to its right represents KCa in the presence of wild-type SLN. A shift to the right indicates a decrease in apparent affinity of SERCA1 for Ca2+. Data are mean ± S.D.; *, p < 0.05 versus SERCA1, and dagger , p < 0.05 versus SERCA1/wild-type SLN coexpression. B, expression of SLN constructs in HEK-293 T-cell microsomes. SERCA1 and SLN, with a FLAG epitope attached at its N terminus (NF-SLN), or mutant NF-SLN constructs, expressed in HEK-293 T-cell microsomes, were separated on Tris/Tricine gels and subjected to immunoblotting with anti-FLAG antibody.

                              
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Table II
Effect of SLN constructs on Ca2+ transport by SERCA1
Effects of SLN on SERCA1 function are expressed in terms of KCa and Vmax for the experiments presented in Fig. 3A. ND, not determined.


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Fig. 4.   Effect of SLN on Ca2+ uptake activity by SERCA1. Ca2+ dependence of Ca2+ uptake was measured for control microsomes expressing SERCA1 (open circles) or microsomes coexpressing both SERCA1 and Ra-SLN (closed circles). The specific activity of SERCA1 was determined by quantitative ELISA, and Ca2+ uptake rates were normalized to Vmax of SERCA1 expressed in absence of SLN. The values are given as mean of the percentage of SERCA1 Vmax ± S.D.

Several constructs were derived from Ra-SLN, and their effects were analyzed by coexpression with rabbit SERCA1. Since attempts to produce antibodies against SLN were not successful in our hands, we attached a FLAG epitope with the sequence MDYKDDDDK to the N terminus of SLN (NF-SLN). The resulting construct shifted KCa to the same extent as Ra-SLN (-0.18 pCa units). Moreover, the Vmax effects of NF-SLN and Ra-SLN were indistinguishable. The N terminus of SLN is not well conserved among different species, suggesting that it is not critical for SLN regulatory function. The fact that attachment of a highly polar tag to the N terminus of SLN did not affect its function provided further evidence for this view.

Sarcoplasmic reticulum vesicles coexpressing SERCA1 and NF-SLN were incubated with anti-FLAG antibody prior to the Ca2+ uptake assay to determine whether the interaction between the two proteins was reversible by antibody. At saturating Ca2+ levels (pCa 5.5) antibody treatment decreased Ca2+ uptake rates of NF-SLN/SERCA1 from 1.48 (140% of SERCA1 control) to 1.15 nmol/min/µg (109% of control), reversing most of the stimulatory effect of SLN on Vmax (Table III). At low Ca2+ concentrations, pCa 6.75, incubation with anti-FLAG antibody enhanced Ca2+ uptake rates from 0.125 (59% of SERCA1 control) to 0.185 nmol/min/µg (87%), thereby reversing the inhibitory effect of SLN on SERCA1 due to a reduction in apparent Ca2+ affinity. Uptake rates of control microsomes expressing only SERCA1 (1.06 at pCa 5.5 and 0.213 nmol/min/µg at pCa 6.75) were not affected by antibody treatment. These experiments confirm that SLN affects both KCa and Vmax of Ca2+ transport by SERCA1.

                              
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Table III
Effect of anti-FLAG antibody on SERCA1 activity regulated by NF-SLN
Microsomes were incubated with anti-FLAG antibody; control microsomes were incubated with boiled antibody, and Ca2+ transport was measured as described under "Materials and Methods." Ca2+ uptake values are given in nmol/min/µg SERCA1 protein, and uptake relative to a SERCA1 control is presented in parentheses as % of control. Data are mean values ± S.D.

We investigated whether Ser4 or Thr5 in the motif Arg-Ser-Thr of Ra-SLN might be phosphorylated, thereby regulating SLN function. We did not observe any phosphorylated SLN with either native sarcoplasmic reticulum vesicles or microsomes from HEK-293 T-cells coexpressing SERCA1 and Ra-SLN when purified protein kinase A or calmodulin kinase II were added to the experiment or when labeling with endogenous kinases was attempted in the presence of [gamma -32P]ATP (not shown). Mutation of Thr5 to Ala led to a slight gain in inhibitory function, manifesting as a further decrease in apparent Ca2+ affinity. This might imply that prevention of phosphorylation at position 5 by mutation improves the inhibitory function of SLN. The mutation also led to loss of the stimulatory effect of SLN on Vmax (Fig. 3 and Table II). We were unable to evaluate the effects of replacement of Thr5 by Glu, to mimic phosphorylation, because this construct was not expressed (Fig. 3B), and pCa of SERCA1 was unaltered (Table II). Thus, our results do not provide any evidence for a role of phosphorylation in regulating SLN function. The fact that the motif, Arg-Ser-Thr, in Ra-SLN is not conserved in human or in mouse SLN makes it unlikely that phosphorylation of Thr5 is a critical feature in SLN regulation of SERCA1.

The five highly conserved amino acids Arg-Ser-Tyr-Gln-Tyr31, forming the C terminus of SLN, protrude into the lumen of the sarcoplasmic reticulum. Adding the polar, positively charged FLAG epitope to the C terminus of SLN (SLN-FC) resulted in a "super inhibitory" effect, decreasing apparent Ca2+ affinity to pCa 5.98 and lowering Vmax at saturating Ca2+ concentrations to 62% compared with SERCA1 control (Table II). Unfortunately, we could not compare SLN-FC and NF-SLN expression levels, because anti-FLAG antibody did not recognize the FLAG epitope attached to the C terminus of SLN (Fig. 3B).

The C-terminal amino acids Tyr-Gln-Tyr were mutated individually to Ala to study their role in SLN function. Mutant Y29A gained inhibitory function relative to NF-SLN at low Ca2+ concentrations. KCa was reduced to pCa 6.06, and the positive Vmax effect was diminished to 109% compared with SERCA1 (Fig. 3 and Table II). The expression of Y29A was comparable to that of NF-SLN, suggesting that the increase in inhibitory function was due to a stronger interaction of SLN with SERCA1. Mutant Q30A had unaltered function, whereas Y31A shifted KCa to a similar extent as wild-type SLN but inhibited Vmax by 30%.

In an attempt to determine whether tyrosine phosphorylation is involved in regulation of SLN function, we incubated rabbit sarcoplasmic reticulum vesicles or HEK-293 T-cell microsomes coexpressing SERCA1 and either Ra-SLN or NF-SLN under a variety of conditions favoring phosphorylation. We were unable to detect any phosphotyrosine in SLN using two different anti-phosphotyrosine antibodies (not shown). We also investigated the possibility that mutation of Tyr29 and Tyr31 to Glu to introduce a negative charge at the position of each of the Tyr residues might mimic tyrosine phosphorylation. Replacement of Tyr29 by Glu resulted in a slight loss in inhibitory function, manifested as a KCa shift of -0.12 pCa units compared with -0.17 pCa units for Ra-SLN. There was, however, a large loss in the stimulatory effect on Vmax. Whereas SERCA1 Vmax in the presence of Ra-SLN is 143% of control, SERCA1 Vmax in the presence of the Y29E mutant was only 74% of control (the effect on Vmax was converted from stimulatory to inhibitory). The Y31E mutant lost most of its ability to lower the apparent affinity of SERCA1 for Ca2+, and its stimulatory effect on Vmax was converted to an inhibitory effect. The double mutant Y29E/Y31E lost all of its inhibitory function on KCa, but its effect on Vmax was not measured. The expression of these mutants was comparable to that of NF-SLN, suggesting that loss of function was due to a loss of interaction of mutant SLN with SERCA1.

The similarity between SLN and PLN peptide sequences and their functional similarity (decrease in apparent affinity of SERCA1 for Ca2+) suggested that the site of interaction with the transmembrane helices of the corresponding Ca2+-ATPase might be the same. Amino acids Leu8, Asn11, and Val19 in SLN, corresponding to amino acids Leu31, Asn34, and Leu42 in PLN (which lose function when mutated to Ala), were mutated to Ala. L8A and N11A lost most of their inhibitory effect on KCa but retained part of their stimulatory effect on Vmax. Mutant V19A retained its inhibitory effect on KCa but lost most of its stimulatory effect on Vmax.

SLN mutants I17A and L21A (corresponding to PLN monomeric, gain of function mutants I40A and L44A) had unaltered KCa effects but lost part of their stimulatory effect on Vmax. SLN mutant V14A, corresponding to PLN monomeric gain of function mutant L37A, had intermediate effects on both KCa and Vmax. We have not found any evidence for a higher oligomeric structure through examination of the mobility of boiled versus unboiled samples of SLN in SDS-PAGE patterns for NF-SLN or purified SLN. When coexpressed in HEK-293 T-cells, SLN did not change the monomer/oligomer ratio of PLN (not shown).

Since our results indicated differences between SLN/SERCA1 interactions and PLN/SERCA2a interactions, we investigated whether the two proteins might act at the same site in SERCA1. PLN suppresses the Ca2+ transport activity of SERCA1 by lowering its apparent affinity for Ca2+ to pCa 6.13 (28). In our experiments, a plateau of inhibitory effect was reached with a PLN/SERCA1 molar ratio of 2:1 in transfection assays. Similarly, a plateau of inhibitory effect was reached with a SLN/SERCA1 molar ratio of 4:1. When both Ra-SLN and PLN were coexpressed with SERCA1 in a ratio of either 4:2:1 or 5:3:1, KCa was shifted to pCa 5.99, and Vmax was increased by 32%, indicating that the effects of Ra-SLN and PLN on Ca2+ transport were additive. This suggests that the two regulatory proteins may interact with SERCA1 at different sites.

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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Fig. 5.   SLN expression in HEK-293 T-cell microsomes and in chronic low frequency stimulated and contralateral rabbit fast-twitch skeletal muscle sarcoplasmic reticulum. Microsomal proteins from HEK-293 T-cells, cotransfected with SERCA1 and Ra-SLN cDNAs, or rabbit fast-twitch skeletal muscle sarcoplasmic reticulum proteins from normal animals, or from animals subjected to chronic low frequency stimulation were separated on Tris/Tricine gels. The gels were stained with Coomassie Blue R250. SLN was purified from transfected HEK-293 T-cell microsomes or from native muscle sarcoplasmic reticulum, as described under "Materials and Methods." A, 1st lane, polypeptide standard, the size of the peptide markers is indicated; 2nd lane, 30 µg of microsomal proteins from transfected HEK-293 T-cells; 3rd lane, Ra-SLN purified from 200 µg of microsomal proteins from transfected HEK-293 T-cells; 4th lane, 30 µg of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum proteins. B, 1st lane, polypeptide standard; 2nd lane, 30 µg of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum proteins; 3rd lane, SLN purified from 200 µg of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum proteins.

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 beta -actin were included as internal controls. We did not attempt to obtain absolute values, but we were able to show that the ratio between normal and control samples for GAPDH levels did not change after stimulation. The values obtained for PCR products of SERCA1, SLN, and beta -actin were then normalized to values obtained for GAPDH (100 ± 19% at 3 days; 100 ± 23% at 4 days) in each muscle sample. SERCA1 mRNA expression did not change after 3 days (101 ± 17%) or 4 days (97 ± 36%) of stimulation. A decrease in SLN mRNA levels to 84 ± 20% at 3 days and 86 ± 24% at 4 days was detected, but the alteration was not statistically significant at p < 0.05. Surprisingly, beta -actin mRNA concentrations dropped to 50 ± 20% at 3 days and 58 ± 17% at 4 days after stimulation.

The slightly reduced expression of SLN mRNA in stimulated muscles was compared at the protein level. Coomassie-stained gels containing a total of 30 µg of separated microsomal proteins were subjected to densitometric analysis. Since SERCA1 protein concentrations did not change after stimulation, we normalized densitometric values of SLN bands to SERCA1 values from the same muscle sample. Comparison of SERCA1/SLN ratios from contralateral normal and stimulated muscle samples revealed a 40 and 38% reduction of SLN protein concentration for 3-day and 4-day stimulated muscles, respectively (Fig. 6). Fig. 6A also shows that a protein at 14 kDa is up-regulated in stimulated muscle sarcoplasmic reticulum. The identity and role of this protein remains to be elucidated.


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Fig. 6.   Effect of chronic low frequency stimulation of rabbit fast-twitch skeletal muscle on SLN expression. Microsomal proteins (30 µg) were separated on Tris/Tricine gels as described in the legend to Fig. 5. A, 30 µg of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum proteins; 1st lane, 3-day stimulated rabbit muscle; 2nd lane, contralateral muscle (3-day control); 3rd lane, 4-day stimulated rabbit muscle; 4th lane, contralateral muscle (4-day control); 5th lane, polypeptide standard. Closed arrows point to the band identified as SLN, and an open arrow points to a 14-kDa band induced in 3-day and 4-day samples. Alternatively, 4 µg of microsomal proteins were applied on SDS gels to obtain separation of SERCA1 (not shown). All gels were stained with Coomassie Blue R250, and SLN and SERCA1 bands were analyzed by scanning densitometry. SERCA1 protein expression, determined both by scanning densitometry and by quantitative ELISA analysis, was unaltered after stimulation (not shown). B, ratios of SLN to SERCA1 were determined for each muscle sample, and values are presented as percentage of contralateral values. Values obtained after stimulation for 3-days are indicated by a white bar, and values from 4-day stimulated animals are indicated by a shaded bar.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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+ of -0.18 pCa units upon coexpression with SLN. This decrease is less pronounced than the decrease observed upon expression of SERCA1 with PLN (-0.32 pCa units, Fig. 3). The decrease in KCa, however, was not the only effect of SLN on SERCA1 function. SLN also stimulates Vmax by about 40%. Therefore, SLN modulates SERCA1 in two ways as follows: at low Ca2+ concentrations SLN inhibits Ca2+ uptake rates due to a decrease in Ca2+ affinity, and at saturating Ca2+ concentrations it stimulates Ca2+ uptake rates by SERCA1 (Fig. 4). Incubation with anti-FLAG antibody reversed both effects of NF-SLN, confirming that NF-SLN affects both KCa and Vmax.

The N-terminal sequence of SLN does not seem to be critical for the observed shift in KCa. Hu-SLN, Ra-SLN, and NF-SLN have significant differences in their N-terminal amino acid sequences. For example, amino acids Glu-Arg-Ser4 in Ra-SLN are replaced by Gly-Ile-Asn4 in Hu-SLN, without alteration of the net charge in the N-terminal sequence. In NF-SLN, the FLAG epitope elongated the Ra-SLN N terminus, and the addition of 7 charged residues altered the net charge of the N terminus from 0 to -3. Nevertheless, Hu-SLN, Ra-SLN, and NF-SLN had identical regulatory effects on SERCA1. In the model of Kimura et al. (32), inhibitory interactions occur in the intramembrane domains of PLN and SERCA2a, and cytosolic interactions can regulate these inhibitory interactions but are not, themselves, inhibitory. In the SLN·SERCA1 complex, cytosolic interactions may not occur at all.

The inhibitory effect of PLN is relieved by phosphorylation at N-terminal residues Ser16 and Thr17 (6). Monomeric PLN is the most inhibitory form of PLN (32), and phosphorylation seems to stabilize PLN pentamer formation (33). We did not detect any oligomeric structure in SLN separated on Tris/Tricine gels and in the presence of 1% SDS, but we cannot exclude the formation of oligomeric structures in SLN under physiologic conditions. Although the calculated molecular mass of SLN is 3733 Da, it migrates with a mobility corresponding to 6 kDa on SDS-PAGE. It is unlikely that the 6-kDa band represents a dimer, because boiling in the presence of 1% SDS did not affect mobility, as it does for PLN (32). We did not detect any phosphorylated SLN in experiments using purified kinases or under conditions favoring phosphorylation by endogenous protein kinases. We attempted to introduce a negative charge into the potential phosphorylation site, RST, by mutation of Thr5 to Glu, but the mutant T5E was poorly expressed and could barely be detected by anti-FLAG antibody, so that its effect on function was not measurable. Mutant T5A gained inhibitory function by lowering KCa and did not stimulate Vmax.

SLN-FC with the FLAG epitope added to the C-terminal end of SLN gained inhibitory function. This observation, together with the effects of mutations of Tyr29 and Tyr31, demonstrated that the C-terminal sequence of SLN could play an important role in SLN regulatory function. The mutant Y29A gained inhibitory function by lowering KCa but lost most of its stimulatory effect on Vmax. Mutants Y29E and Y31E retained part of their inhibitory effect on KCa but lost part of their stimulatory effect on Vmax. We were unable to detect any phosphotyrosine in SLN using [gamma -32P]ATP and autoradiography or using ATP and two different anti-phosphotyrosine antibodies. Nevertheless, we can predict from our studies of the Y29E and Y31E mutants (Table II) that the introduction of a negative charge at the C terminus of SLN by phosphorylation, should it occur, would be likely to diminish both regulatory effects of SLN on Ca2+ transport by SERCA1. Ca2+ affinity of SERCA1 would be predicted to be increased, but Vmax would be predicted to be decreased. It is of interest, however, that elongation of the C terminus with an eight amino acid sequence containing a net negative charge of -3 (SLN-FC) decreased Vmax and decreased Ca2+ affinity dramatically, thereby increasing the inhibitory function of SLN. The mechanism of this gain of SLN inhibitory function is not understood.

Although SLN stimulates the Vmax of SERCA1, we have been unable to observe a comparable stimulation of Vmax by the interaction of PLN with SERCA1. Expression of SERCA1 in HEK-293 T-cells, in the presence or absence of PLN, did not result in any differences in Vmax of Ca2+ uptake rates by SERCA1, although a decrease in KCa of -0.31 pCa units was observed (Table II). These findings confirm previous studies that demonstrated that PLN does not affect Vmax of Ca2+ uptake rates by SERCA2a (17, 34, 35).

Kimura et al. (32) presented a model for the reversible inhibition of SERCA2a activity by PLN, in which interactions between monomeric PLN and SERCA2a in the transmembrane domains are inhibitory, and interactions between the two proteins in cytosolic domains can modulate the transmembrane inhibitory interactions through long range coupling. Interaction can be reversed, however, by phosphorylation of the N-terminal domain of PLN or by binding of Ca2+ to the transmembrane domain of SERCA2a. Alanine-scanning mutagenesis provided evidence for two functional domains on opposite faces of the PLN transmembrane helix. Mutations in one face reduced inhibitory interactions with SERCA2a and did not change PLN monomer/pentamer ratio. This helical face was proposed to be in contact with amino acids in SERCA2a. Mutations in the opposite helical face increased PLN monomer concentration and enhanced inhibitory interactions, leading to the conclusion that the PLN monomer is the regulatory species.

The transmembrane helices of SLN and PLN share considerable sequence homology. In 19 transmembrane amino acids, 8 are identical, 7 share Val, Leu, or Ile, 3 share Thr, Trp, or Cys, and one is a Met for Ile substitution. There is, however, no evidence that SLN forms an oligomeric structure. We mutated SLN residues corresponding to key PLN residues in either the SERCA2a interaction face or the PLN interaction face. SLN mutants L8A and N11A, corresponding to loss of function mutants in PLN, lost their ability to decrease apparent Ca2+ affinity but retained part of their ability to stimulate Vmax. In contrast, mutant V19A retained its effect on KCa but lost most of its stimulatory effect on Vmax. Analysis of mutants I17A and V19A, corresponding to monomeric, gain of function mutants in PLN, revealed unaltered effects on KCa but reduced effects on Vmax. Mutant V14A showed intermediate effects on KCa and Vmax.

Thus, the pattern of effects of mutations in SLN does not reproduce the pattern of effects of mutations in PLN. The fact that SLN not only affects KCa but also stimulates Vmax, together with the differences observed in the mutational analysis of SLN and PLN, suggests that the mechanisms of interaction of SLN and PLN with the transmembrane helices of the Ca2+-ATPase may be different. The effects of SLN and PLN on KCa of SERCA1 were additive when both proteins were coexpressed with SERCA1 in HEK-293 T-cells under conditions where inhibition by one or the other of the two inhibitors alone had plateaued. This suggests that SLN and PLN interact at different sites in SERCA1 molecules and that the regulatory mechanisms are different. Nevertheless, since the transmembrane helices of SERCA1 and SERCA2a are highly conserved, SLN and PLN retain the potential to modulate the activity of both SERCA1 and SERCA2a. Indeed, the ectopic expression of PLN in fast-twitch muscle in transgenic mice is associated with a decrease in the apparent affinity of SERCA1 for Ca2+ (36). Muscle relaxation was prolonged in transgenic mice compared with wild-type animals, demonstrating that PLN is able to inhibit SERCA1 activity and decrease muscle relaxation rates in the presence of SLN expressed in vivo.

Role 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 beta -actin mRNA and the up-regulation of a 14-kDa protein in the sarcoplasmic reticulum of stimulated muscles represent early adaptations to low frequency stimulation of protein expression. In the time-dependent process of fast-to-slow fiber type transition, myofibrillar and Ca2+ regulatory protein isoforms are converted from fast-twitch isoforms to their slow-twitch isoform counterparts. In this process the SERCA1·SLN complex which is regulated through both KCa and Vmax effects is replaced by a SERCA2a·PLN complex with powerful KCa regulation but no Vmax regulation (46, 47).

Navarre et al. (48) discovered that two low molecular weight proteolipids are associated with the yeast H+-ATPase. In double mutants lacking both proteolipids, the Vmax of the H+-ATPase was reduced to 50% that of wild-type strains (49). The yeast proteolipids do not bear any obvious homology to PLN or SLN, and the mechanism of their interaction with the H+-ATPase has not been investigated in detail.

Our studies have uncovered a number of interesting features concerning the interaction of SLN with SERCA1, which can be compared and contrasted with the effects of PLN on SERCA2. Like PLN, SLN is a regulator of SERCA function. Both SLN and PLN have inhibitory effects on KCa, but, in contrast to PLN, SLN has clear stimulatory effects on Vmax. Like PLN, SLN inhibitory effects on KCa are overcome by elevation of Ca2+ concentration, suggesting that transmembrane interactions are inhibitory. Unlike PLN, SLN stimulates Vmax at saturating Ca2+ concentrations. In contrast to PLN, SLN regulation does not appear to be modulated by phosphorylation of a cytoplasmic domain. In this respect, our proposal that PLN/SERCA2a interactions are regulated through a 4-base circuit (32) is not likely to hold for SLN/SERCA1a interactions. The two regulatory molecules share transmembrane sequence homology, and both are able to regulate SERCA1 and SERCA2 in identical fashion, presumably because SERCA1 and SERCA2 have such high sequence identity in critical transmembrane helices. Nevertheless, SLN and PLN do not appear to bind to identical sequences in SERCA1, since the inhibitory effects of PLN and SLN are additive. SLN probably provides a constant stimulation of SERCA1 Vmax, since we have not observed any mechanism for rapid switching to or from the stimulatory mode. We have, however, provided evidence that SERCA1 activity might be modulated over the long term by the level of SLN expression.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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