Sarcolipin Protein Interaction with Sarco(endo)plasmic Reticulum Ca2+ATPase (SERCA) Is Distinct from Phospholamban Protein, and Only Sarcolipin Can Promote Uncoupling of the SERCA Pump*

Background: Sarcolipin and phospholamban, the regulators of SERCA, are differentially expressed in muscle. Results: Only sarcolipin binds to SERCA in the presence of Ca2+ and interacts with SERCA throughout the kinetic cycle. Conclusion: Sarcolipin alone promotes uncoupling of the SERCA pump leading to increased heat production. Significance: Sarcolipin-mediated regulation of SERCA plays an important role in muscle-based thermogenesis. Sarco(endo)plasmic reticulum Ca2+ATPase (SERCA) pump activity is modulated by phospholamban (PLB) and sarcolipin (SLN) in cardiac and skeletal muscle. Recent data suggest that SLN could play a role in muscle thermogenesis by promoting uncoupling of the SERCA pump (Lee, A.G. (2002) Curr. Opin. Struct. Biol. 12, 547–554 and Bal, N. C., Maurya, S. K., Sopariwala, D. H., Sahoo, S. K., Gupta, S. C., Shaikh, S. A., Pant, M., Rowland, L. A., Bombardier, E., Goonasekera, S. A., Tupling, A. R., Molkentin, J. D., and Periasamy, M. (2012) Nat. Med. 18, 1575–1579), but the mechanistic details are unknown. To better define how binding of SLN to SERCA promotes uncoupling of SERCA, we compared SLN and SERCA1 interaction with that of PLB in detail. The homo-bifunctional cross-linker (1,6-bismaleimidohexane) was employed to detect dynamic protein interaction during the SERCA cycle. Our studies reveal that SLN differs significantly from PLB: 1) SLN primarily affects the Vmax of SERCA-mediated Ca2+ uptake but not the pump affinity for Ca2+; 2) SLN can bind to SERCA in the presence of high Ca2+, but PLB can only interact to the ATP-bound Ca2+-free E2 state; and 3) unlike PLB, SLN interacts with SERCA throughout the kinetic cycle and promotes uncoupling of the SERCA pump. Using SERCA transmembrane mutants, we additionally show that PLB and SLN can bind to the same groove but interact with a different set of residues on SERCA. These data collectively suggest that SLN is functionally distinct from PLB; its ability to interact with SERCA in the presence of Ca2+ causes uncoupling of the SERCA pump and increased heat production.

The sarco(endo)plasmic reticulum Ca 2ϩ ATPase (SERCA) 3 is primarily responsible for maintaining low cytosolic and high luminal Ca 2ϩ in the sarcoplasmic reticulum (SR) of muscle by coupling energy from ATP hydrolysis to transport Ca 2ϩ (1,55). SERCA pump activity is modulated by phospholamban (PLB) and sarcolipin (SLN) in cardiac and skeletal muscle (2). Although PLB and SLN have been considered to be homologous proteins, there are several distinct features that have been overlooked. First, the pattern of expression of these proteins is very distinct. In mammals, PLB is expressed in cardiac muscle and to a lesser extent in slow twitch skeletal muscles, whereas SLN is predominantly expressed in skeletal muscles and its expression in the heart is restricted to atria (3,4). Interestingly, SLN expression is severalfold higher in fast and slow twitch skeletal muscles of larger mammals when compared with rodents (3). Secondly, they bear key structural differences both at the N terminus and at the C terminus with limited sequence similarity in the transmembrane region (5)(6)(7)(8)(9). The transmembrane helix of SLN is only 19 amino acids (aa) long, whereas that of PLB is 30 aa long with 21 aa within the membrane and 9 aa protruding out into the cytosol (supplemental Fig. 1) (8). The cytosolic portion of PLB has an additional helix, whereas the unstructured cytosolic SLN region allows flexibility. Further, only SLN has a luminal segment (5 aa) that protrudes into the SR lumen, whereas PLB does not.
These distinct properties of SLN and PLB suggest that they play unique roles in cardiac and skeletal muscle physiology. The function of PLB has been well studied; PLB in the dephosphorylated state is known to decrease the apparent affinity of Ca 2ϩ without any effect on the V max of SERCA (10,11). The inhibitory effect of PLB on SERCA is abolished at high Ca 2ϩ and PLB phosphorylation at Ser-16 or Thr-17 by protein kinase A and Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII) as a result of ␤-adrenergic stimulation (8,10,11). The interaction between PLB and SERCA has been characterized extensively by chemical cross-linking and co-immunoprecipitation experiments (12)(13)(14)(15)(16)(17)(18)(19)(20). Recent studies have shown that the presence of Ca 2ϩ influences SERCA-PLB as well as SERCA-SLN interac-tions. PLB binds most favorably to the Ca 2ϩ -free E2 state of SERCA in the presence of ATP, but cannot functionally interact with SERCA at high Ca 2ϩ . These studies concluded that PLB and Ca 2ϩ binding to SERCA are mutually exclusive (15,18,19,21). In contrast, the mechanism of SLN inhibition of SERCA is poorly understood. Previous studies have suggested that SLN can affect the affinity and/or the V max of SERCA pump (22)(23)(24)(25).
It is currently unknown whether SLN binds to SERCA in the same region as PLB and whether SLN binding to SERCA has a different functional outcome when compared with PLB (12,26,27). Mall et al. (28) showed that SLN binding to SERCA promotes uncoupling of the SERCA pump and slippage of Ca 2ϩ into the cytoplasm instead of the SR lumen. These studies also suggested that SLN binding with SERCA can increase ATP hydrolysis and heat production and therefore could contribute to muscle thermogenesis (29,30). We further tested this idea in vivo by generating an SLN Ϫ/Ϫ mouse model (22,31). Our results showed that SLN is essential for thermogenesis in muscle and that mice lacking SLN develop hypothermia when exposed to acute cold. Moreover SLN Ϫ/Ϫ mice became significantly obese when fed on high fat diet, whereas WT mice were less obese and significantly up-regulated SLN expression (31). These data suggested that the muscle-based SLN-SERCA interaction contributes to heat production and energy expenditure.
A major goal of this study was to investigate how SLN binding with SERCA contributes to muscle thermogenesis. In the current study, we investigated whether PLB plays a role in muscle thermogenesis using PLB Ϫ/Ϫ mice. Our results show that PLB was not essential for thermogenesis. By comparing SLN binding with SERCA with that of PLB-SERCA, we demonstrate that SLN-SERCA interaction is unique; it can bind to SERCA in the presence of high Ca 2ϩ and interact with SERCA throughout the kinetic cycle, which may facilitate uncoupling of SERCA. Using mutagenesis, we show that SLN binds with a different set of residues on SERCA when compared with PLB. These findings provide new insight into the molecular basis of SLN-SERCA interaction and highlight that SLN alone is responsible for muscle thermogenesis.
Mouse Models and Acute Cold Challenge Experiments-PLB Ϫ/Ϫ mice were a kind gift from Litsa Kranias, University of Cincinnati. SLN Ϫ/Ϫ mice have been generated previously (22). Both PLB Ϫ/Ϫ and SLN Ϫ/Ϫ mice were bred (C57BL/6J genetic background) and housed at ambient temperature. The study protocol was approved by the Ohio State University Institutional Animal Care and Use Committee (OSU-IACUC). Acute cold exposure of mice to 4°C was performed in the Comprehensive Lab Animal Monitoring System (CLAMS) setup as described before (31).
Mutagenesis and Expression of SLN, PLB, and SERCA in HEK293 Cells-The rat SERCA1 cDNA sequence, which has 99% homology with mouse SERCA1, was cloned into the pcDNA3.1 (ϩ) vector. Mouse SLN and PLB cDNAs were PCRamplified and cloned into pcDNA3.1 (ϩ) vector. Desired mutagenesis of rat SERCA1, mouse SLN, and mouse PLB were done using the QuikChange TM site-directed mutagenesis kit (Agilent Technologies). All cDNA clones and mutated constructs were confirmed by direct sequencing. HEK293 cells were co-transfected with SERCA and SLN or PLB construct cDNAs using Lipofectamine 2000. Co-expression was done at 1:2 ratios of SERCA and SLN or PLB. 48 h after transfection, cells were harvested in PBS, and pellet was stored at Ϫ80°C after flash freezing in liquid nitrogen. Microsomes from transfected cells were prepared as described previously (32). Briefly, cells were resuspended in a hypotonic solution containing 10 mM Tris⅐HCl (pH 7.5) and 0.5 mM MgCl 2 for 20 min. Protease inhibitor was added, and cells were homogenized by 30 strokes in a Dounce homogenizer on ice. Homogenates were diluted by an equal volume of 10 mM Tris⅐HCl (pH 7.5), 0.5 M sucrose, 300 mM KCl. The cell extracts were centrifuged at 10,000 ϫ g to pellet cell debris. The supernatants were diluted with KCl to a final concentration of 0.6 M and centrifuged at 100,000 ϫ g for 1 h at 4°C. The pellet was resuspended in storage buffer containing 10 mM MOPS (pH 7.0) and 10% sucrose and stored at Ϫ80°C in small aliquots.
Chemical Cross-linking of SLN to SERCA-Chemical crosslinking of proteins was performed by homo-bifunctional sulfhydryl cross-linker BMH (18). SERCA and SLN or PLB were cross-linked in cross-linking buffer containing 40 mM MOPS (pH 7.0), 3.2 mM MgCl 2 , 75 mM KCl, and 1 mM EGTA. 15 g of microsomes was mixed in cross-linking buffer, and 3 mM ATP was added followed by the addition of 0.1 mM cross-linker to start cross-linking. The reaction was incubated for 1 h at 25°C. The reaction was stopped by the addition of SDS-PAGE sample-loading buffer containing 100 mM dithiothreitol. Specific cross-linking of SLN to SERCA interaction shows a 113-kDa band probed with anti-SLN antibody, whereas PLB interaction with SERCA shows a 116-kDa band probed with anti-PLB antibody.
Effect of Ca 2ϩ and TG on SERCA and SLN Interaction-The effect of Ca 2ϩ on SLN binding was assessed by the addition of increasing concentrations of Ca 2ϩ in the presence of ATP and cross-linker as described previously (31). The effect of TG, a SERCA inhibitor, was studied by adding TG (0 -10 M) to the reaction mixture without Ca 2ϩ , in the presence of ATP before the addition of cross-linker. The reactions were stopped by adding SDS sample buffer and analyzed as described previously.
SERCA-mediated Ca 2ϩ Uptake and ATP Hydrolysis Assays-To determine the inhibitory effect of SLN on SERCA-mediated Ca 2ϩ transport, oxalate-supported Ca 2ϩ uptake assay was performed (33). SERCA1 was co-transfected with pcDNA3.1, SLN, or E7C-SLN in HEK cells. Briefly, HEK homogenates were incubated in buffer containing 20 mM MOPS (pH 7.0), 5 mM MgCl 2 , 100 mM KCl, 5 mM NaN 3 , 5 mM ATP, 5 mM K ϩ -oxalate, and 0.5 mM EGTA. Different concentrations of CaCl 2 were added to obtain desired free Ca 2ϩ levels as determined by the MAXCHELATOR program. Samples were incubated in reaction buffer, and aliquots were collected at different time intervals and filtered through 0.45-m filters. The filters were washed by wash solution, and bound radioactive Ca 2ϩ was measured by scintillation counting. ATPase activities were measured using the BIOMOL green phosphate assay (28,34). Microsomes were incubated in the Ca 2ϩ uptake buffer containing 15-20 g of protein in the presence or absence of 5 M ionophore (A23187). The reaction was initiated by the addition of CaCl 2 , and samples were collected at different time intervals. The amount of P i release was calculated as nmol P i /mg/min. Control reactions were carried out in the presence of TG.
Cross-linking of SLN to the Various Kinetic States of SERCA-The major intermediates of SERCA kinetic steps and their stable analogs were produced by incubating microsomes in crosslinking reaction buffer with different analogs for 45 min at 25°C before the addition of BMH (35,36). The E2 state was obtained by incubating the microsomes without ATP. E1⅐Ca 2 and E1PCa 2 reaction tubes contained 100 M free Ca 2ϩ in the absence and presence, respectively, of 3 mM ATP. E1⅐AlF x ⅐ADP complex, an E1PCa 2 ⅐ADP analog, was obtained by incubating microsomes with 50 M AlCl 3 , 3 mM KF, and 3 mM ADP. E2⅐AlF 4 Ϫ , the transition state analog of the E2P hydrolysis, was produced by the addition of 50 M AlCl 3 and 3 mM KF to cross-linking buffer. E2V i , the E2P analog, was produced by preincubating microsomes with 0.1 mM orthovanadate. Chemical cross-linking was done by adding 0.1 mM BMH at 25°C for 1 h. Cross-linked samples were analyzed by SDS-PAGE and immunoblotted with anti-SLN antibody or anti-PLB antibody.

PLB Does Not Play a Role in Muscle
Thermogenesis-PLB and SLN are key regulators of the SERCA pump, but it is currently unknown whether PLB is also important for thermogenesis as found for SLN (31). Therefore, in this study, PLB Ϫ/Ϫ and SLN Ϫ/Ϫ mice were challenged to acute cold (4°C), and their core body temperature (T c ) was followed for a period of 8 h. To minimize contribution from brown adipose tissue (BAT), we also surgically removed interscapular BAT (iBAT) from one set of mice. Results showed that after 8 h of cold exposure, SLN Ϫ/Ϫ mice (with iBAT) had a reduced T c (34.0 Ϯ 0.3°C). Moreover iBAT-ablated SLN Ϫ/Ϫ mice showed a further decrease in T c (28.2 Ϯ 1.5°C) and developed severe hypothermia, as reported previously (31). However, both PLB Ϫ/Ϫ and WT mice (with or without iBAT) showed similar heat generation capacity during cold challenge and were able to maintain optimal T c at ϳ37°C, suggesting that the absence of PLB does not affect thermogenesis. These data clearly demonstrate that PLB is not involved in heat generation and that SLN alone is responsible for muscle thermogenesis (Fig. 1A).
SLN Decreases SERCA Ca 2ϩ Uptake (V max ) but Does Not Affect ATP Hydrolysis-The finding that SLN alone is responsible for muscle thermogenesis prompted us to further define how SLN interacts with SERCA and modulates pump activity in detail. Previous studies have reported opposing results; some studies showed that SLN causes a decrease in Ca 2ϩ affinity, whereas others showed an increase or decrease in V max of Ca 2ϩ uptake (13, 22-25, 27, 28, 37). To further characterize the effect of SLN on SERCA, both Ca 2ϩ uptake and ATP hydrolysis assays were performed using microsomes from HEK cells transfected with SERCA1 alone or with SLN. The expression level of SERCA and SLN was verified by immunoblotting using specific antibodies (supplemental Fig. 2). Oxalate-supported Ca 2ϩ uptake assay showed that the presence of SLN did not affect the apparent Ca 2ϩ affinity of SERCA1 (EC 50 SERCA ϭ 6.5 Ϯ 0.1; EC 50 SERCA ϩ SLN ϭ 6.4 Ϯ 0.09 mean Ϯ S.E.). In contrast, SLN inhibited the V max of the SERCA pump at all Ca 2ϩ concentrations (Fig. 1B). Our result showed a 33% decrease in maximal Ca 2ϩ uptake, and this finding that SLN primarily inhibits the V max of SERCA is very similar to previously reported results in mouse hearts deficient in SLN and in studies of rat slow twitch muscle with SLN gene transfer (22,24). Next, we determined whether SLN had an effect on ATP hydrolysis by measuring the ATPase activity at different Ca 2ϩ concentrations (Fig.  1C). This was also tested both in the presence and in the absence of Ca 2ϩ ionophore (A23187) because the addition of ionophore dissipates the Ca 2ϩ concentration gradient across the SR membrane and abolishes the back inhibition of SERCA activity (38). Quantitation of phosphate release showed that there was no significant difference in ATP hydrolysis between samples expressing SERCA alone or with SLN (Fig. 1C). As expected, the addition of ionophore increased the ATPase activity in both samples; however, the presence of SLN did not further modify ATPase activity.
SLN Binds to SERCA in the Same Groove as PLB-Next, we sought to map the site of SLN binding on SERCA. SLN and SERCA1 were expressed in HEK cells, and protein-protein interaction was studied using a 10 Å chemical cross-linker. The structural basis for PLB and SERCA interaction has been well characterized in vitro using different length chemical crosslinkers (15,16,18,19). These studies have shown that residue Asn-30 of PLB resides less than a distance of 10 Å from cysteine 318 of SERCA2, when the proteins functionally interact (15,18). To identify the SLN residues that lie at/or within 10 Å from Cys-318 of SERCA, selected amino acids in SLN were mutated to cysteine. The SLN mutants studied include S4C-SLN (corresponding to N27C-PLB), E7C-SLN (corresponding to N30C-PLB), L8C-SLN and F9C-SLN ( Fig. 2A), and V26C-SLN (corre-sponding to V49C of PLB that cross-links with V89C-SERCA) (15). The binding between SERCA1 and SLN was studied with homo-bifunctional cross-linking reagent BMH (Fig. 2, A and  B), and the cross-linked SLN-SERCA complex was detected by immunoblotting with anti-SLN antibody. Data showed that residues S4C and E7C in SLN could be specifically cross-linked to Cys-318 of SERCA1, whereas L8C and F9C in SLN could not ( Fig. 2A), indicating that Ser-4 and Glu-7 of SLN lie at/within 10 Å from Cys-318 of SERCA. Similarly the C-terminal V26C-SLN showed specific cross-linking with V89C-SERCA1 in the presence of BMH, suggesting that Val-26 of SLN and Val-89 of SERCA are located within 10 Å distance (Fig. 2B). These results suggest that SLN and PLB bind to the same groove of SERCA formed by TMs M2, M4, M6, and M9 (13).
SLN Interacts with SERCA in the Presence of High Ca 2ϩ , but Its Interaction Is Abolished by Thapsigargin-It is well known that Ca 2ϩ modulates the activity of SERCA, and at high Ca 2ϩ , SERCA is maximally activated. We therefore studied the effect of Ca 2ϩ on SERCA-SLN binding and compared how SLN binding differs from that of PLB-SERCA at similar Ca 2ϩ concentrations. SLN can be maximally cross-linked to SERCA in the absence of Ca 2ϩ . Interestingly, as shown in Fig. 2C, SLN continues to interact with SERCA even at high Ca 2ϩ (100 M), but at a reduced level, both at the N terminus (E7C) and at the C terminus (V26C) cross-linking sites. In contrast, PLB binding with SERCA is abolished at Ca 2ϩ concentrations above 1 M (Fig. 2C). These data suggest that unlike PLB, SLN is able to bind to SERCA even in the presence of high Ca 2ϩ (concentrations ranging from 5 to 100 M), which is an important distinction between these two molecules. To prove that the binding between SLN and SERCA is dynamic and requires an active SERCA pump, we investigated the effect of TG, a known inhibitor, which forms an irreversible and nonphysiological complex with SERCA (39,40). We examined the effect of increasing concentrations of TG (1-10 M) and found that at a higher concentration (10 M), TG completely abolished the binding between SERCA and SLN at both the N terminus and the C terminus (Fig. 2D). TG also had a similar inhibition on PLB-SERCA binding (Fig. 2D).
PLB and SLN Interact with Different Sets of Transmembrane Residues in SERCA-It has been shown that point mutations in the transmembrane helices M2, M4, and M6 of SERCA1 affect the interaction between SERCA1 and PLB (13,41). To determine whether SLN binds to the same transmembrane residues as PLB, the transmembrane residues were mutated to alanine. The SERCA1 transmembrane mutants (V89C, L321A, V795A, L802A, T805A, and F809A) were co-expressed together with either E7C-SLN or N30C-PLB in HEK cells. Our cross-linking studies showed that SLN binding with SERCA1 is not significantly affected by any of these mutations except F809A (Fig.  3A). On the other hand, mutations L321A, V795A, L802A, T805A, and F809A in SERCA significantly decrease PLB binding with SERCA (Fig. 3, A and B) as has been reported previously (13,41). These results suggest that although they bind to the same groove on SERCA, the binding sites for SLN and PLB are not identical.
Only SLN Can Bind to Various Kinetic States of SERCA Pump-Our finding that SLN decreases the V max of Ca 2ϩ uptake and competitively binds to SERCA in the presence of high Ca 2ϩ (Figs. 1 and 2) indicated that SLN may be affecting one or more kinetic steps during the SERCA reaction cycle. Recent studies also suggested that SLN binding to SERCA could promote uncoupling of Ca 2ϩ transport from the ATP hydrolysis activity of the SERCA pump resulting in heat production (28,30). To investigate whether SLN can bind to the various kinetic steps of SERCA during the catalytic cycle, we chemically induced various SERCA transition steps (kinetic isomers) by exposing SERCA to metal fluorides and vanadate and performed crosslinking (35,36,42,43). Our results show that SLN can be crosslinked to SERCA in the Ca 2ϩ -free E2 state, and the addition of ATP significantly increases the interaction between SERCA and SLN (Fig. 3C). Binding of both ATP and Ca 2ϩ transitions SERCA to E1⅐Ca 2 and E1PCa 2 as a result of ATP hydrolysis (43). We found that SLN is able to interact with SERCA at these kinetic states. As shown in Fig. 2C, SLN also interacts to the Ca 2ϩ -free E2P state induced by E2V i or E2⅐AlF 4 Ϫ , suggesting that SLN occupies SERCA during the whole catalytic cycle. PLB interaction with SERCA was further investigated using the same chemical modifications; it was found that PLB also interacts with the Ca 2ϩ -free E2 form of SERCA and that binding of ATP further enhances the interaction as reported previously (12,15,18). The binding of Ca 2ϩ to ATP-bound SERCA abolished the interaction between PLB and SERCA. PLB is also unable to bind the subsequent phospho-intermediates, E1PCa 2 and E2P of the catalytic cycle (Fig. 3D). Surprisingly, even the Ca 2ϩ -free phospho-intermediate (E2P) did not interact with PLB. Importantly, these studies suggest that SLN functions very differently from PLB; it can interact with the SERCA intermediates tested here, and its ability to remain bound during the catalytic cycle may facilitate uncoupling of the pump from Ca 2ϩ transport.
SLN Can Interact with SERCA Even after Both Ca 2ϩ Binding Sites Are Occupied-SERCA has two Ca 2ϩ binding sites, I and II, located adjacent to each other, surrounded by transmembrane helices M4, M5, M6, and M8 (44 -46). These sites show cooperativity in Ca 2ϩ binding, and site II can bind the second Ca 2ϩ ion only when site I is filled by the first Ca 2ϩ ion, to trigger the kinetic cycle of the SERCA pump in the presence of ATP. Therefore, the site I mutant (E771Q) precludes any Ca 2ϩ binding to SERCA pump, whereas the site II mutant (E309Q) is able to bind one Ca 2ϩ ion at site I (21,42,44). To determine how Ca 2ϩ binding to each of these sites in SERCA affects the ability of SLN to interact with SERCA, we mutated site I (E771Q) and site II (E309Q) individually as reported earlier (21,44). D351A-SERCA mutant, which binds Ca 2ϩ at both sites as well as ATP but is unable to hydrolyze the ATP, was further utilized to test whether SLN binds to Ca 2ϩ -bound SERCA (47). The data shown here demonstrate that only SLN can bind to SERCA that is already bound to Ca 2ϩ and ATP (Fig. 4). This is evident from the results showing that D351A-SERCA interacts with SLN, but not PLB, at all Ca 2ϩ concentrations tested. Interestingly, PLB binding with D351A-SERCA is abolished at a low Ca 2ϩ concentration of 0.1 M. We also observed that SERCA with site I mutation (E771Q) binds with SLN as well as PLB under all the Ca 2ϩ concentrations tested. Cross-linking of SLN to either of these SERCA mutants (E771Q or E309Q) showed that loss of Ca 2ϩ binding sites increased the level of SLN interaction with SERCA even at high Ca 2ϩ , in contrast to SLN binding to WT SERCA (Fig. 4A). On the other hand, PLB was able to bind to the site I mutant (E771Q SERCA) at all Ca 2ϩ concentrations, whereas when site II was mutated (E309Q SERCA), PLB interaction with SERCA could be detected even at 5 M Ca 2ϩ but was competed out at higher Ca 2ϩ (Fig. 4B) (21). SLN, however, continues to bind E309Q SERCA even at high Ca 2ϩ (100 M). These findings provide direct evidence that SLN binding to SERCA is distinct from PLB and that its ability to interact with Ca 2ϩ -bound SERCA can promote uncoupling of the pump.

DISCUSSION
Using both gain of function and loss of function of SLN mouse models, we recently demonstrated that SLN plays a unique role in muscle physiology and that the SLN-SERCA interaction is an important contributor to muscle-based thermogenesis (31). Our goals in this study were two-fold: 1) to determine whether PLB plays a role in muscle thermogenesis and 2) to understand how the SLN interaction with SERCA differs from that of PLB and the basis for SLN-mediated SERCA uncoupling and muscle thermogenesis. Our studies showed that PLB is not essential for thermogenesis. Therefore, we focused our efforts on defining the uniqueness of SLN-SERCA interaction by comparing it with that of PLB-SERCA. We chose to employ the chemical cross-linking strategy over co-immunoprecipitation (13) because co-immunoprecipitation requires solubilization and disruption of the SR membrane architecture, which will destroy the native interaction between SERCA and SLN. Moreover cross-linking agents have been shown to be reliable reagents not only for deciphering accurate distances between key residues of interacting protein molecules, but also for monitoring the dynamic changes between protein molecules that affect the protein-protein interactions (15,18,19,21).
Data from this study reveal that SLN interaction with SERCA differs significantly from PLB; SLN can bind to SERCA even at high Ca 2ϩ ( up to 100 M), and it remains bound to SERCA during the SERCA kinetic cycle, whereas PLB does not bind to SERCA at high Ca 2ϩ (above 1 M) or bind to SERCA kinetic intermediate states. A notable finding of our study is that the presence of SLN significantly decreases the V max of Ca 2ϩ uptake; however, ATP hydrolysis is unaffected. Our results are in agreement with recent studies showing that SLN has no effect on ATPase activity at saturating concentrations of Ca 2ϩ in unsealed or sealed membrane preparations (28,30). Collectively, our data showing that despite inhibition of V max , ATP hydrolysis is unchanged, suggest that in the presence of SLN, SERCA continues to hydrolyze ATP but less Ca 2ϩ is transported to the lumen of the SR, thus implicating SLN as an uncoupler of SERCA (30). PLB, on the other hand, decreases SERCA Ca 2ϩ transport and ATP hydrolysis only at lower Ca 2ϩ concentrations, but has no effect at higher Ca 2ϩ concentrations (47)(48)(49) Previous modeling studies have shown that the PLB interaction site lies in a groove on the surface of SERCA formed by TMs M2, M4, M6, and M9 (13). Using mutagenesis in the SERCA TM regions and co-immunoprecipitation assays, Asahi et al. (13) showed that PLB and SLN bind to the same groove of SERCA and that the same set of amino acids interacts with SLN or PLB. Our studies showed that SLN can be cross-linked to SERCA at Cys-318 and Val-89 in SERCA as found for PLB, indicating that they bind to the same groove on SERCA. Moreover our studies employing SERCA TM mutants suggested that except for mutation F809A, other mutations including L321A and L802A had no effect on cross-linking between the two molecules (Fig. 3, A and B). Interestingly, although the L802A-SERCA1 mutation did not interfere with SLN binding to SERCA, it completely abolished the interaction of PLB with SERCA (Fig. 3, A and B). These and other published results suggest that SLN and PLB may bind to the same groove in SERCA (26,50); however, our studies suggest that they may not interact with the same set of amino acids in the TM domain of SERCA. In addition, differences in the N terminus of the two molecules may determine whether it can remain bound to Ca 2ϩ -bound SERCA. On the other hand, the protruding C terminus of SLN composed of five unique residues (RSYQY) could provide additional points of interaction for stronger binding with SERCA (51). A detailed understanding of SLN contact points with SERCA requires additional structural studies.
An important goal of this study was to determine how the SLN interaction with SERCA differs from PLB during the Ca 2ϩ transport cycle. Following Ca 2ϩ binding, SERCA hydrolyzes ATP and transitions into E1PCa 2ϩ , and subsequently to E2P, before releasing Ca 2ϩ into the lumen (46,(52)(53)(54). We found that SLN is able to interact with different SERCA phosphointermediates during the kinetic cycle (Fig. 3C), whereas PLB can only interact with the Ca 2ϩ -free E2 state. Further, our data showing that Ca 2ϩ effectively competes out PLB, but not the SLN interaction with SERCA, suggest that SLN has a higher affinity to SERCA. These novel findings suggest that SLN alone can promote the uncoupling of SERCA ATPase activity from Ca 2ϩ transport. Thus SLN binding to SERCA prevents release of Ca 2ϩ into the lumen by promoting slippage of Ca 2ϩ back to the cytosol (Fig. 5).
We also studied how Ca 2ϩ binding influences SLN interaction with SERCA using mutagenesis. During Ca 2ϩ transport, sequential binding of Ca 2ϩ to sites I and II promotes conformational changes resulting in occlusion of Ca 2ϩ in the presence of ATP (42,43). Our mutagenesis studies of Ca 2ϩ binding sites I and II indicate that Ca 2ϩ binding to SERCA modifies the ability of PLB and SLN to bind to SERCA. When site I is mutated, SERCA remains in a Ca 2ϩ -free (E2) state, allowing maximal binding of SLN or PLB. However, mutation of site II allows Ca 2ϩ binding to site I (requires higher Ca 2ϩ ) and abolishes PLB interaction (21). The finding that SLN continues to bind to the site II mutant further confirms that SLN can bind to Ca 2ϩbound SERCA. These data, along with the data showing that SLN interacts with SERCA throughout the kinetic cycle, further support that SLN alone can promote uncoupling of SERCA.
This study reveals important insights on how SLN differs from PLB. Our data conclusively show that 1) SLN interacts with SERCA in the presence of high Ca 2ϩ , 2) only SLN remains bound to SERCA throughout the kinetic cycle, and 3) PLB cannot interact with Ca 2ϩ -bound SERCA. Based on our findings, we propose a model to illustrate how SLN binding leads to uncoupling of the SERCA pump in Fig. 5. The model shows that SLN and SERCA are co-localized in the SR and that their functional interaction is modulated by Ca 2ϩ . In the absence of Ca 2ϩ , SLN interacts maximally with the E2 state of SERCA. When the Ca 2ϩ level is increased, the majority of the SLN is released from SERCA, but a fraction of the pumps remains bound to SLN. SLN binding to SERCA allows ATP hydrolysis to proceed but interferes with Ca 2ϩ transport into SR, instead promoting release of Ca 2ϩ back to the cytosol (54).
These studies additionally suggest how PLB and SLN regulation of SERCA can produce distinct physiological outcomes in muscle. PLB is predominant in the heart, and its ability to modulate Ca 2ϩ affinity in a phosphorylation-dependent manner is well suited to regulate cardiac function during rest and exercise, requiring different rates of Ca 2ϩ transport. SLN, on the other hand, does not affect pump affinity for Ca 2ϩ but primarily affects V max by causing inefficiency and promoting uncoupling of SERCA contributing to enhanced heat production. FIGURE 5. Schematic diagram depicting SLN uncoupling of SERCA. Ca 2ϩbound SERCA is shown in green, and the Ca 2ϩ -free form is shown in blue. The functional interaction between SLN (shown in red) and SERCA is shown as dotted bridges. SLN binds strongly to ATP-bound SERCA in the E2 state and remains bound during Ca 2ϩ binding to sites I and II. Ca 2ϩ binding activates ATP hydrolysis and transition to the E1P Ca 2ϩ . The continued presence of SLN inhibits normal transition from E1P to E2P and promotes the premature release of Ca 2ϩ to the cytosol. Following slippage of Ca 2ϩ , SLN loses its affinity for SERCA, and the pump transitions to the E2P and E2P i states. The subsequent release of P i returns SERCA to the E2 state. The model predicts that both SLN and Ca 2ϩ bind simultaneously to SERCA but that the presence of SLN prevents luminal opening of SERCA.