Sarcolipin is a conformation-dependent regulator of the sarcoplasmic reticulum calcium pump SERCA

Sarcolipin is a conformation-dependent regulator of the sarcoplasmic reticulum calcium pump SERCA | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sarcolipin is a conformation-dependent regulator of the sarcoplasmic reticulum calcium pump SERCA Joseph O. Primeau, M. Joanne Lemieux, Paul LaPointe, Howard S. Young This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9544726/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Muscle relaxation is enabled by the sarco-endoplasmic reticulum Ca 2+ - ATPase (SERCA), which removes calcium from the cytosol and returns it to the lumen of the sarcoplasmic reticulum. During transport, SERCA adopts a variety of conformational states which differ in their structure and affinity for substrates. The transition between these states is mediated by calcium and ATP binding and the formation of an aspartyl-phosphate intermediate, which enables the transport of calcium across the membrane. SERCA function is highly regulated because of the importance of calcium in processes such as muscle contraction-relaxation. A family of tissue-specific transmembrane regulatory subunits interact with SERCA, exemplified by sarcolipin (SLN) in skeletal muscle and phospholamban (PLN) in cardiac muscle. SLN and PLN are known to alter the apparent calcium affinity and maximal activity of SERC. In the present study, we investigated SLN inhibition of SERCA under conditions that varied the substrate-dependent conformational state of SERCA. Measuring both calcium-dependent ATP hydrolysis and charge translocation, we found that SLN inhibition was dependent on the initial state of SERCA. Under substrate conditions that poised SERCA in the calcium-free E2 state, SLN was more inhibitory and impacted both the maximal activity and apparent calcium affinity of SERCA. In contrast, SLN inhibition was reduced under pre-incubation conditions that favored the calcium-bound E1 state of SERCA. We conclude that SLN is capable of distinct modes of interaction with SERCA depending on the conformational state, and that the mode of interaction exhibits conformational memory in that the initial state persists during steady-state turnover of SERCA. Sarcoplasmic reticulum calcium transport charge translocation ATPase activity P-type ATPase regulatory peptide Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The sarco-endoplasmic reticulum calcium pump (SERCA) maintains the low resting-state calcium levels in the cytosol that are essential for many physiological signaling processes. A well-characterized example is the relaxation phase of the contraction-relaxation cycle in muscle. Following calcium release and muscle contraction, SERCA uses the energy of ATP to transport calcium ions into the sarcoplasmic reticulum (SR) lumen to achieve muscle relaxation. The calcium transport mechanism of SERCA has been extensively studied (e.g. (Sorensen, Moller & Nissen, 2004 ; Toyoshima & Mizutani, 2004 ; Toyoshima et al., 2000 ; Toyoshima & Nomura, 2002 )). SERCA progresses through a series of intermediates that alternate between high-affinity (E1) and low-affinity (E2) states for calcium. Calcium transport requires the binding of ATP (E2·ATP) and the cooperative binding of two calcium ions (E1·ATP·Ca) to form a catalytically competent conformation of SERCA. The energy of ATP is captured in the form of a phospho-enzyme intermediate (E1 ~ P·Ca) and released through a conformational change that facilitates calcium translocation (E2-P). Following calcium release into the SR lumen, the phosphate is hydrolyzed and released, returning SERCA to a low-affinity state (E2). From this point, SERCA is poised to begin the transport cycle anew. It is important to note that the presence or absence of substrates can be used to poise SERCA in different conformational states (e.g. E2, E2·ATP, E1·Ca) and that the crystal structures of these conformational states have been determined. SERCA activity is regulated by several tissue-specific transmembrane peptides called the regulins, which include phospholamban (PLN) and dwarf open reading frame (DWORF) in cardiac muscle and sarcolipin (SLN) and myoregulin (MLN) in skeletal muscle (Anderson et al., 2015 ; Anderson et al., 2016 ; Nelson et al., 2016 ). SLN is a 31-residue, α-helical integral membrane protein, consisting of a short cytoplasmic domain that contains a regulatory phosphorylation site, a transmembrane domain, and a highly conserved luminal tail. Like PLN, SLN has been reported to reduce the apparent calcium affinity of SERCA (Gorski et al., 2013 ; Odermatt et al., 1998 ) by interacting with SERCA in an inhibitory groove formed by transmembrane helices M2, M6, and M9. Crystal structures revealed binding of SLN to the inhibitory groove of SERCA, which stabilized a calcium-free E1-like intermediate of SERCA that appears poised between the calcium-free E2 and calcium-bound E1 states (Toyoshima et al., 2013 ; Winther et al., 2013 ). The transmembrane portion of SLN mediates this interaction; however, SLN regulation is strongly dependent on the unique and highly conserved luminal Arg 27 -Ser-Tyr-Gln-Tyr 31 domain of SLN. While SLN was thought to be functionally homologous to PLN, the mechanism by which SLN regulates SERCA is distinct (Gorski et al., 2013 ). In addition to SLN’s role in regulating calcium homeostasis in skeletal muscle, it has been suggested that SLN also plays a role in thermogenesis and energy metabolism (Bal et al., 2012 ). SLN was reported to uncouple SERCA ATPase activity from calcium transport by promoting the backflow of calcium into the cytosol during ATP hydrolysis. A mechanism for SLN-mediated uncoupling through the interaction of SLN’s N-terminus with SERCA has been put forward (Autry, Thomas & Espinoza-Fonseca, 2016 ). Given that skeletal muscle accounts for approximately 40% of adult body mass, the energy lost from SERCA uncoupling would make a significant contribution to heat generation and thermogenesis. This unique physiological role of SLN suggests a unique SERCA regulatory mechanism. The crystal structures of SERCA in the presence of SLN revealed a unexpected conformation of SERCA – an E1-like, calcium-free state that appears poised between the E2 and E1 states (Toyoshima et al., 2013 ; Winther et al., 2013 ). These structures are very similar to the crystal structure of SERCA-PLN complex (Akin et al., 2013 ). While these structures provide a plausible mechanism for SERCA inhibition, they yield little insight into the functional differences between PLN and SLN and the potential uncoupling behavior of SLN. In our previous work, we reported that the conformational state of SERCA influenced the interaction with PLN, suggesting that multiple conformational states of the SERCA-PLN complex were possible. Moreover, once a particular complex was engaged, the functional effect persisted through multiple SERCA turnover events of the catalytic cycle, and we termed this process “conformational memory” (Smeazzetto et al., 2017 ). Herein, we performed a similar study of SERCA regulation by SLN. Through the use of particular substrates, SERCA was poised in the calcium-free E2 state (pre-incubation in the absence of substrates), the calcium-bound E1 state (pre-incubation with calcium), ATP-bound E2 state (pre-incubation with ATP), and a physiological resting state (pre-incubation with ATP & resting calcium). Proteoliposomes containing SERCA in the absence and presence of SLN were pre-incubated under these different substrate conditions. Following pre-incubation, calcium-dependent ATPase activity and charge translocation were measured. SLN decreased the maximal activity of SERCA under all substrate conditions tested. In contrast, SLN altered the apparent calcium affinity of SERCA only under one condition, pre-incubation in the absence of substrates. There was no evidence of SERCA uncoupling in the SERCA-SLN proteoliposomes. Nonetheless, the results indicated two possible SERCA-SLN regulatory complexes that depend on the conformation of SERCA. Once formed, these conformations are stable through steady-state turnover of SERCA. While both SERCA-PLN (Smeazzetto et al., 2017 ) and SERCA-SLN complexes exhibited conformational memory, the results support the concept that SLN is a distinct regulatory subunit of SERCA. Results The co-reconstituted proteoliposomes used in this study approximate the SR membrane in protein density, orientation, and lipid composition (Ceholski, Trieber & Young, 2012 ; Gorski et al., 2013 ; Trieber, Afara & Young, 2009 ; Trieber et al., 2005 ; Young et al., 1997 ). The proteoliposomes are unilamellar, SERCA molecules are oriented in the membrane with the cytoplasmic domains accessible to substrates on the outer surface (Young et al., 1997 ), and the lipid-to-protein ratio is ~ 120 to 1 (Trieber et al., 2005 ). These proteoliposome characteristics mimic native skeletal muscle SR membranes, and they allow precise measurement of steady-state calcium-dependent ATPase activity (e.g. (Ceholski et al., 2012 ; Gorski et al., 2013 ; Trieber et al., 2009 )) and pre-steady state calcium transport (Smeazzetto et al., 2017 ). SERCA ATPase activity and calcium transport The proteoliposomes containing SERCA in the absence and presence of SLN were pre-incubated under four different substrate conditions: (i) pre-incubation in the absence of substrates (substrate jump condition), (ii) pre-incubation with calcium (ATP jump condition), (iii) pre-incubation with ATP (calcium jump condition), and (iv) pre-incubation under physiological conditions (80 nM calcium, 4 mM ATP; physiological calcium jump condition). Following pre-incubation under these substrate conditions, we measured calcium-dependent ATP hydrolysis and calcium transport by SERCA (Fig. 1B, C ). ATP hydrolysis was measured as a function of calcium concentration (0.1–10 µM free calcium). The data were fit to the Hill equation and the kinetic parameters maximal activity (V max ) and apparent calcium affinity (K Ca ) were determined from the resultant curve fits (Fig. 2). The kinetic parameters obtained under various starting conditions are summarized in Table 1 . Charge translocation by SERCA was also measured as a function of calcium concentration using a surface electrogenic even reader (SEER; SURFE 2 R N1, Nanion Technologies GmbH). Proteoliposomes containing SERCA in the absence and presence of SLN were adsorbed to a solid supported membrane (SSM) on the surface of a gold electrode as previously described (Smeazzetto et al., 2017 ; Tadini-Buoninsegni, 2020 ) (Fig. 1C). The current transients observed following pre-incubation and initiation of the reaction were analyzed for peak current (nA) and total charge translocation (nC). The data were plotted as peak current (Fig. 3) and total charge translocation (Fig. 4) versus calcium concentration. These data were then fit to the Hill equation and the kinetic parameters maximal activity (V max ) and apparent calcium affinity (K Ca ) were determined from the resultant curve fits (Table 1 ). Peak current measurements resulted in statistically significant differences in the maximal activity (V max ) of SERCA, and charge translocation measurements resulted in statistically significant differences in the apparent calcium affinity (K Ca ) of SERCA. Overall, the trends observed with ATPase activity measurements (Fig. 2) were consistent with the charge translocation measurements (Figs. 3 & 4). Table 1 Kinetic parameters for SERCA and SERCA-SLN proteoliposomes derived from steady-state ATPase activity measurements and pre-steady state charge transfer measurements. Starting conditions Kinetic parameters K Ca (µM) V max (µmoles/min/mg) (nA) (nC) n H Substrate jump Pre-incubate in the absence of substrates; start reaction with 4 mM ATP and 0.1 to 10 µM Ca 2+ SERCA ATPase−activity 0.42 ± 0.02 4.1 ± 0.1 1.5 ± 0.1 SERCA-SLN ATPase−activity 0.97 ± 0.04 2.8 ± 0.1 1.6 ± 0.1 SERCA Peak−current 1.00 ± 0.10 3.9 ± 0.3 1.5 ± 0.1 SERCA-SLN Peak−current 1.30 ± 0.10 2.1 ± 0.3 1.3 ± 0.1 SERCA Charge−translocation 0.53 ± 0.04 0.80 ± 0.05 1.6 ± 0.1 SERCA-SLN Charge−translocation 0.94 ± 0.09 0.70 ± 0.09 1.5 ± 0.1 ATP jump Pre-incubate with 0.1 to 10 µM Ca 2+ ; start reaction with 4 mM ATP SERCA ATPase−activity 0.27 ± 0.02 3.0 ± 0.1 1.1 ± 0.1 SERCA-SLN ATPase−activity 0.45 ± 0.07 2.5 ± 0.1 1.0 ± 0.1 SERCA Peak−current 0.83 ± 0.03 4.8 ± 0.5 2.0 ± 0.1 SERCA-SLN Peak−current 1.04 ± 0.06 3.7 ± 0.7 1.6 ± 0.1 SERCA Charge−translocation 0.48 ± 0.02 0.90 ± 0.10 1.7 ± 0.1 SERCA-SLN Charge−translocation 0.61 ± 0.03 0.70 ± 0.10 1.6 ± 0.1 Calcium jump Pre-incubate with 4 mM ATP; start reaction with 0.1 to 10 µM Ca 2+ SERCA ATPase−activity 0.35 ± 0.02 3.9 ± 0.1 1.5 ± 0.1 SERCA-SLN ATPase−activity 0.37 ± 0.03 2.3 ± 0.1 1.6 ± 0.1 SERCA Peak−current 1.18 ± 0.05 3.8 ± 0.3 1.3 ± 0.1 SERCA-SLN Peak−current 1.40 ± 0.10 1.6 ± 0.3 1.1 ± 0.1 SERCA Charge−translocation 0.65 ± 0.02 0.84 ± 0.06 1.5 ± 0.1 SERCA-SLN Charge−translocation 0.98 ± 0.08 0.46 ± 0.06 1.3 ± 0.1 Physiological calcium jump Pre-incubate with 4 mM ATP and 80 nM Ca 2+ ; start reaction with remaining Ca 2+ SERCA ATPase−activity 0.37 ± 0.01 3.5 ± 0.1 1.5 ± 0.1 SERCA-SLN ATPase−activity 0.46 ± 0.03 3.0 ± 0.1 2.0 ± 0.2 SERCA Peak−current 0.86 ± 0.03 3.5 ± 0.3 1.5 ± 0.1 SERCA-SLN Peak−current 0.91 ± 0.06 2.9 ± 0.4 1.6 ± 0.1 SERCA Charge−translocation 0.47 ± 0.03 0.61 ± 0.06 1.7 ± 0.1 SERCA-SLN Charge−translocation 0.59 ± 0.03 0.55 ± 0.07 1.7 ± 0.1 Values shaded in grey indicated SERCA and SERCA-SLN comparisons that were statistically significant (p > 0.01). Substrate jump condition Proteoliposomes containing SERCA in the absence and presence of SLN were pre-incubated in the absence of substrates. This pre-incubation condition favors the E2 state of SERCA, which is a low-affinity state for calcium binding. The substrate jump condition involved the simultaneous addition of calcium and ATP to initiate ATPase activity (Fig. 2 & Table 1 ). As previously observed (Gorski et al., 2013 ), SLN altered both the maximal activity (V max ) and apparent calcium affinity (K Ca ) of SERCA. Proteoliposomes containing SERCA alone yielded a V max of 4.1 ± 0.1 µmol/min/mg, while proteoliposomes containing SERCA and SLN yielded a V max of 2.8 ± 0.1 µmol/min/mg. Similarly, proteoliposomes containing SERCA alone yielded a K Ca value of 0.42 ± 0.02 µM calcium, compared to 0.97 ± 0.04 µM calcium for proteoliposomes containing SERCA and SLN. The increase in K Ca indicates a decrease in the apparent calcium affinity of SERCA in the presence of SLN, which is a characteristic feature of SERCA inhibition. Thus, SLN decreased both the maximal activity and apparent calcium affinity of SERCA under these conditions. The substrate jump condition was next used to activate calcium transport by SERCA-containing proteoliposomes adsorbed to an SSM. Peak currents (nA) and total charge translocation (nC) were measured for current traces over a range of calcium concentrations. Proteoliposomes containing SERCA alone yielded a V max of 3.9 ± 0.3 nA, while proteoliposomes containing SERCA and SLN yielded a V max of 2.1 ± 0.3 nA (Fig. 3). Similarly, the K Ca values were found to be 0.53 ± 0.04 and 0.94 ± 0.09 µM calcium for SERCA in the absence and presence of SLN, respectively (Fig. 4). Thus, the V max and K Ca values determined under these conditions using SEER technology were comparable to the values obtained from the calcium-dependent ATPase activity measurements (Table 1 ). ATP jump condition Proteoliposomes containing SERCA in the absence and presence of SLN were next pre-incubated with calcium concentrations ranging from 0.1 to 10 µM before initiating SERCA ATPase activity (ATP jump; Fig. 2 & Table 1 ). This pre-incubation condition favors the E1 state of SERCA, which is a high-affinity state for calcium binding. Compared to the substrate jump condition, the overall impact of SLN on SERCA activity was significantly reduced. The maximal activity of SERCA was lower with a modest depression in the presence of SLN (V max values of 3.0 ± 0.1 µmol/min/mg for SERCA alone compared to 2.5 ± 0.1 µmol/min/mg for SERCA-SLN). In addition, the effect of SLN on the apparent calcium affinity (K Ca ) of SERCA was more modest, and the observed K Ca values were shifted to higher affinity (0.27 ± 0.02 µM calcium for SERCA alone compared to 0.45 ± 0.07 µM calcium for SERCA-SLN). Consequently, pre-incubation of SERCA proteoliposomes with calcium resulted in a higher-affinity, slower pump, which manifested as an increase in the apparent calcium affinity of SERCA (decreased value for K Ca ) and a decrease in the maximal activity of SERCA. SLN inhibition of SERCA was still evident, but considerably diminished by pre-incubation with calcium. The ATP jump condition was used to activate calcium transport by SERCA-containing proteoliposomes adsorbed to an SSM using SEER technology. The measured charge translocation compared well with the ATPase activity measurements. Proteoliposomes containing SERCA alone yielded a V max of 4.8 ± 0.5 nA, while proteoliposomes containing SERCA and SLN yielded a V max of 3.7 ± 0.7 nA (Fig. 3). While there was an apparent reduction in V max , these values were not statistically different. K Ca was found to be 0.48 ± 0.02 and 0.61 ± 0.03 µM calcium for SERCA in the absence and presence of SLN, respectively (Fig. 4). Thus, inhibition of SERCA calcium transport by SLN was also diminished following pre-incubation of SERCA in the presence of calcium (Table 1 ). Calcium jump condition The proteoliposomes containing SERCA in the absence and presence of SLN were pre-incubated with 4 mM ATP (absence of calcium), and SERCA ATPase activity was initiated by the addition of calcium (calcium jump condition; Fig. 2 & Table 1 ). This pre-incubation condition favors the ATP-bound, calcium-free E2 state of SERCA. The V max values were determined to be 3.9 ± 0.1 µmol/min/mg for SERCA alone compared to 2.3 ± 0.1 µmol/min/mg for SERCA in the presence of SLN. The V max values observed for pre-incubation with ATP were comparable to the substrate jump condition. In contrast, the effect of SLN on the apparent calcium affinity of SERCA was not observed with ATP pre-incubation (K Ca of 0.35 ± 0.02 µM calcium for SERCA alone compared to 0.37 ± 0.03 µM calcium for SERCA-SLN). These measurements indicated that ATP pre-incubation preserved the effect of SLN on the V max of SERCA. However, there was no change in the apparent calcium affinity of SERCA, indicating that this aspect of SLN inhibition was lost in the E2·ATP state of SERCA. The calcium jump condition was next used to activate calcium transport by SERCA-containing proteoliposomes. SERCA alone yielded a V max of 3.8 ± 0.3 nA, while proteoliposomes containing SERCA and SLN yielded a V max of 1.6 ± 0.3 nA (Fig. 3). This was the largest decrease in SERCA V max observed in the presence of SLN. K Ca was found to be 0.65 ± 0.02 and 0.98 ± 0.08 µM calcium for SERCA in the absence and presence of SLN, respectively (Fig. 4). Thus, for the ATPase activity and charge translocation measurements, the primary effect of SLN on SERCA under ATP pre-incubation conditions was depression of SERCA’s V max (Table 1 ). Physiological calcium jump The different pre-incubation conditions described above poised SERCA in well-known conformational states – E2, E1, and E2·ATP, respectively. However, these pre-incubation conditions differ from the physiological resting state (mM ATP and nM calcium). To mimic the physiological resting state, SERCA and SERCA-SLN proteoliposomes were pre-incubated with 4 mM ATP and 80 nM calcium, near the resting level of cytosolic calcium that myocytes experience. The ATPase activity of SERCA and SERCA-SLN proteoliposomes under these conditions was determined (physiological calcium jump condition; Fig. 2 & Table 1 ). The observed V max depression remained between SERCA and SERCA-SLN proteoliposomes, though it was reduced (V max values of 3.5 ± 0.1 and 3.0 ± 0.1 µmol/min/mg, respectively). The K Ca shift observed between SERCA and SERCA-SLN proteoliposomes was also reduced (K Ca values of 0.37 ± 0.01 and 0.46 ± 0.03 µM calcium, respectively). This small change in the apparent calcium affinity of SERCA was akin to the weak SLN inhibition observed for the calcium pre-incubation (ATP jump) and ATP pre-incubation (calcium jump) conditions. These trends in the presence of SLN – V max depression and weak SERCA inhibition – agreed with previous observations for SERCA in the presence of PLN (Smeazzetto et al., 2017 ). The physiological calcium jump condition was next used to activate calcium transport by SERCA-containing proteoliposomes. Similar to the ATPase activity measurements for this pre-incubation condition, the effects of SLN on the V max (3.5 ± 0.3 and 2.9 ± 0.4 nA respectively; Fig. 3 ) and K Ca (0.47 ± 0.03 and 0.59 ± 0.03 µM calcium, respectively; Fig. 4 ) of SERCA were reduced. Thus, the primary effect of SLN on SERCA under physiological pre-incubation conditions was depression of SERCA’s V max (Table 1 ). These data are consistent with the ATPase activity of SERCA in the absence and presence of SLN (Fig. 2 & Table 1 ). Summary of Results SLN consistently decreased the maximal activity (V max ) of SERCA under all conditions tested, using both ATPase activity and charge translocation as measures of SERCA function. This effect was more pronounced under calcium-free pre-incubation conditions that promoted the E2 state of SERCA (Figs. 2 & 3; substrate jump & calcium jump conditions) and diminished under calcium pre-incubation conditions that promoted the E1 state of SERCA (Figs. 2 & 4; ATP jump & physiological calcium jump conditions). SLN altered the apparent calcium affinity of SERCA (K Ca ) under one condition (Table 1 ; substrate jump condition), though there were minor changes in K Ca under most other conditions. Comparative charge translocation by SERCA, SERCA-SLN, and SERCA-PLN SLN has been reported to uncouple SERCA calcium transport from ATP hydrolysis as a means of heat production in non-shivering thermogenesis (Bal et al., 2012 ; Mall et al., 2006 ; Sahoo et al., 2015 ). This is considered a unique property of SLN regulation of SERCA in skeletal muscle, and not a property of PLN regulation of SERCA in cardiac muscle. To investigate the possible uncoupling of calcium transport from ATP hydrolysis, we compared the ATP hydrolysis rates (V max in Table 2 ) with the total charge translocation for SERCA in the absence and presence of SLN ( Figure S1 ). The underlying assumption was that uncoupling should manifest as a selective reduction in charge movement for SERCA in the presence of SLN. We also compared the total charge translocation by SERCA in the presence of SLN to SERCA in the presence of PLN (Fig. 5). No differences were observed in the kinetic parameters derived from total charge translocation (Table 2 ), suggesting that calcium transport is similar in SERCA-SLN and SERCA-PLN proteoliposomes. In addition, under most pre-incubation conditions, the maximal activity of SERCA in ATPase activity measurements (V max ; Table 2 ) was higher for SERCA-PLN compared to SERCA-SLN. This suggests that coupling of ATP-hydrolysis to charge-translocation was higher for SERCA-PLN and lower for SERCA-SLN. Since we would expect a signature feature of SERCA uncoupling to involve higher ATP hydrolysis and lower calcium transport, this is the opposite trend expected for uncoupling of SERCA by SLN. While there was no evidence of SERCA uncoupling in the SERCA-SLN proteoliposomes, there were advantages and disadvantages in using this membrane reconstitution system. The SERCA-SLN proteoliposomes allowed detailed functional measurements and mechanistic insights in a highly purified system, though they lacked the complexity of the cell and additional factors that may modulate cellular calcium homeostasis. The combination of calcium-dependent ATPase activity and charge translocation measurements allowed comparison of both ATP hydrolysis and calcium transport by SERCA. However, the charge translocation measurements were not quantitative in terms of the number of transported calcium ions or the number of SERCA molecules contributing to the signal. To overcome this, we compared the relative charge translocation by SERCA, SERCA-SLN, and SERCA-PLN proteoliposomes. Table 2 Kinetic parameters for SERCA-SLN and SERCA-PLN proteoliposomes derived from steady-state ATPase activity measurements and pre-steady state charge transfer measurements (total charge translocation). Starting conditions Kinetic parameters K Ca (µM) V max (nC) n H Substrate jump Pre-incubate in the absence of substrates; start reaction with 4 mM ATP and 0.1 to 10 µM Ca 2+ SERCA-SLN ATPase−activity 0.97 ± 0.04 2.8 ± 0.1 1.6 ± 0.1 SERCA-PLN ATPase−activity 0.89 ± 0.04 5.9 ± 0.1 2.0 ± 0.1 SERCA-SLN Charge−translocation 0.94 ± 0.09 0.70 ± 0.09 1.5 ± 0.1 SERCA-PLN Charge−translocation 1.02 ± 0.06 0.95 ± 0.02 1.3 ± 0.1 ATP jump Pre-incubate with 0.1 to 10 µM Ca 2+ ; start reaction with 4 mM ATP SERCA-SLN ATPase−activity 0.45 ± 0.07 2.5 ± 0.1 1.0 ± 0.1 SERCA-PLN ATPase−activity 0.82 ± 0.01 3.9 ± 0.1 1.4 ± 0.1 SERCA-SLN Charge−translocation 0.61 ± 0.03 0.70 ± 0.10 1.6 ± 0.1 SERCA-PLN Charge−translocation 0.49 ± 0.03 0.62 ± 0.01 1.8 ± 0.2 Calcium jump Pre-incubate with 4 mM ATP; start reaction with 0.1 to 10 µM Ca 2+ SERCA-SLN ATPase−activity 0.37 ± 0.03 2.3 ± 0.1 1.6 ± 0.1 SERCA-PLN ATPase−activity 0.84 ± 0.03 5.1 ± 0.1 1.3 ± 0.2 SERCA-SLN Charge−translocation 0.98 ± 0.08 0.46 ± 0.06 1.3 ± 0.1 SERCA-PLN Charge−translocation 0.96 ± 0.05 0.54 ± 0.01 1.7 ± 0.1 Physiological calcium jump Pre-incubate with 4 mM ATP and 80 nM Ca 2+ ; start reaction with remaining Ca 2+ SERCA-SLN ATPase−activity 0.46 ± 0.03 3.0 ± 0.1 2.0 ± 0.2 SERCA-PLN ATPase−activity 0.65 ± 0.03 2.8 ± 0.1 3.0 ± 0.4 SERCA-SLN Charge−translocation 0.59 ± 0.03 0.55 ± 0.07 1.7 ± 0.1 SERCA-PLN Charge−translocation 0.66 ± 0.03 0.59 ± 0.01 2.1 ± 0.2 DISCUSSION The pre-incubation conditions described above poised SERCA in two main conformational states: calcium-free E2 and calcium-bound E1. The calcium-free E2 state was promoted by the substrate jump condition (pre-incubation in the absence of substrates) and the calcium jump condition (pre-incubation in the presence of ATP). The calcium-bound E1 state was promoted by the ATP jump condition (pre-incubation in the presence of calcium) and the physiological calcium jump condition (pre-incubation in resting-state conditions). The E2 substrate jump condition is our typical method for initiating functional measurements of SERCA in the presence of SLN (Gorski et al., 2013 ). Under this condition, SERCA and SLN are pre-incubated in the absence of calcium and ATP, and the simultaneous addition of substrates initiated SERCA ATPase activity and calcium transport. As a result, the inhibition of SERCA by SLN manifested as a decrease in both the apparent calcium affinity and maximal activity of SERCA (Table 1 ). The decrease in the apparent calcium affinity of SERCA in the presence of SLN is consistent with previous observations by others (Odermatt et al., 1998 ); however, this effect of SLN was only observed for the substrate jump condition. The primary effect of SLN under all conditions was a depression of SERCA’s maximal activity. This effect was more pronounced for the E2 states of SERCA and diminished for the E1 states of SERCA. Thus, the interaction of SLN with SERCA exhibited a clear preference for the calcium-free E2 states of SERCA. The physiological jump condition pre-incubated SERCA under resting-state physiological conditions followed by a calcium jump to elicit SERCA ATPase activity and charge translocation. Under this pre-incubation condition, SERCA slowly progresses through the calcium transport cycle. The turnover number was approximately 11 per minute (0.1 µmoles/min/mg at 25˚C), while SERCA’s maximum turnover rate in proteoliposomes was ~ 450 per minute for SERCA alone (4.1 µmoles/min/mg at 25˚C). The slow turnover of SERCA through the calcium transport cycle presumably allowed SLN to interact with any preferred conformational state. That said, SERCA may preferentially occupy states that precede the rate-limiting steps in the reaction cycle, particularly the E1 ~ P to E2P transition. Thus, the E1 ~ P conformation of SERCA was a likely candidate for SLN interaction. Consistent with this notion, SERCA regulation by SLN was most similar to the calcium pre-incubation condition (ATP jump; Tables 1 ). We categorized this as a condition that promoted the calcium-bound E1 state of SERCA, which could explain the diminished effect of SLN on SERCA activity (Fig. 2, 3, &, 4 ). SERCA apparent calcium affinity and maximal activity Under most conditions tested, only a slight shift in the apparent calcium affinity of SERCA was observed in the presence of SLN (Table 1 ). Surprisingly, the presence of substrates – calcium, ATP, or both – resulted in a weak effect of SLN on the apparent calcium affinity of SERCA. The exception was pre-incubation of SERCA in the absence of substrates, which resulted in a more inhibitory interaction of SLN and a significant shift in the apparent calcium affinity of SERCA (substrate jump condition; Table 1 ). These results suggested that the physical interaction between SERCA and SLN was sensitive to the conformational state of SERCA and that at least two distinct SERCA-SLN complexes exist. One complex caused a decrease in the maximal activity of SERCA. This was most pronounced for the complex of SLN with the calcium-free E2 states of SERCA, but clearly present under all SERCA conformations tested. A second complex resulted in a decrease in both the apparent calcium affinity and maximal activity of SERCA. These results were unique for the complex of SLN with the substrate-free conformation of SERCA (E2). Surprisingly, this was not observed for the complex of SLN with the calcium-free, ATP-bound conformation (E2·ATP) of SERCA. In a previous study of the SERCA-PLN complex (Smeazzetto et al., 2017 ), we reached a similar conclusion that the calcium-free E2·ATP state of SERCA had distinct structural and functional properties from the substrate-free E2 state of SERCA. This was a surprising finding given that the structures of SERCA in the E2 and E2·ATP states are similar (e.g. compare PDB 1IWO with 2C88 or 3FGO, respectively). SERCA-SLN complexes The results described above point toward two distinct conformational states for the SERCA-SLN complex. One conformation resulted in weaker inhibition of SERCA with minor changes in SERCA kinetics (pre-incubation with calcium in the ATP jump & physiological jump conditions). A second conformation resulted in potent inhibition of SERCA with large changes in SERCA kinetics (pre-incubation without calcium in the substrate jump & calcium jump conditions). We suggest that the weaker inhibitory state corresponds to the known crystal structure of the SERCA-SLN complex (Toyoshima et al., 2013 ; Winther et al., 2013 ), and the potent inhibitory state corresponds to the previous model of the SERCA-PLN complex (Seidel et al., 2008 ; Toyoshima et al., 2003 ). One conformation must correspond to the crystal structure of the SERCA-SLN complex (Toyoshima et al., 2013 ; Winther et al., 2013 ), where SERCA is found in a calcium-free E1-like state (Fig. 6A, B ). This calcium-free E1-like state is closer to the calcium-bound E1 state of SERCA than it is to the calcium-free E2 state. Nonetheless, SLN interacts with M2, M6, and M9 of SERCA to form an inhibitory complex. The nature of the complex suggests that SLN slows transmembrane domain movements that allow SERCA to progress through the calcium transport cycle. However, as SERCA progresses through the transport cycle, SLN must alternately populate two interaction sites – one site that is inhibitory and a second site that is non-inhibitory and releases SERCA for progression through the transport cycle. In the transition from the calcium-free E1-like state to the calcium-bound E1 state of SERCA, the upward movement of transmembrane segment M2 is a major structural change. Side-chain interactions mediate the SLN complex with M2, and only a small change in the position of SLN is required to disengage. Since this complex is poised closer to the calcium-bound state of SERCA, it appears consistent with the weaker inhibitory effect of SLN under some SERCA pre-incubation conditions (ATP jump and physiological jump conditions, which pre-incubated SERCA in the presence of calcium). We suggest that the second conformation corresponds to the calcium-free E2 state of SERCA, as predicted by the original molecular models for the SERCA-PLN complex (Gustavsson et al., 2013 ; Seidel et al., 2008 ; Toyoshima et al., 2003 ) (Fig. 6C, D ). The two conformations, the E1-like state and the E2 state, differ in how SLN is oriented in the inhibitory groove of SERCA formed by transmembrane segments M2, M6, and M9 (Fig. 6E, F & inset ). The binding groove is shallow (partially closed) in the E1-like state of SERCA and deeper (fully open) in the E2 state. This allows SLN to penetrate further into the inhibitory groove in E2, which results in a 5–7 Å lateral translation of SLN compared to the E1-like complex. The position of SLN in the E2 complex partially occupies the position M2 will take in the transition to the calcium-bound E1 state of SERCA. While SLN needs to relocate to allow the movement of M2, the repositioning of SLN required in the E2 complex is larger compared to the E1-like complex. Since this complex is poised further from the calcium-bound state of SERCA and SLN partially occupies the position M2 will take, it appears consistent with the potent inhibitory effect of SLN under some SERCA pre-incubation conditions. These include the substrate jump and calcium jump conditions, which pre-incubated SERCA in the absence of calcium and promoted the calcium-free E2 state. Thus, these conditions seemed consistent with an E2 SERCA-SLN complex that has a potent inhibitory effect on SERCA. Conformational memory The concept of conformational memory has been explored in protein conformational dynamics, inherently disordered proteins, amyloids, and chemical reactions (Csermely et al., 2020 ; Maeda, Taketsugu & Morokuma, 2012 ; Makarov, 2021 ; Schorner et al., 2015 ; Tian, Meng & Zhang, 2020 ). Several proteins exhibit conformational memory by retaining information about past conformations or interactions that influence future activity, e.g. the retention of a ligand bound state after ligand dissociation (Csermely et al., 2020 ; Tripathi, Uversky & Giuliani, 2025 ). SERCA is a molecular machine that undergoes a wide range of conformational states during the transport cycle, all of which require dynamics and flexibility in domain motions. The transitions between these different conformational states of SERCA have been assumed to include spontaneous conformational fluctuations in a stochastic process. However, the availability of substrates can influence the energy landscape for conformational fluctuations and shift from a stochastic reaction coordinate to one with reduced degrees of freedom between SERCA intermediates (conformational memory). The same argument can be made for the interaction of SLN with SERCA. The reaction coordinate between SERCA inhibition by SLN and SERCA’s progression through the calcium transport cycle is not stochastic. We suggest that SLN cycles between inhibitory and non-inhibitory interactions with SERCA, and that these interactions are linked by conformational memory – past SERCA-SLN interactions influence future transport cycles (Fig. 7). It is important to remember that SLN remains associated with SERCA during the catalytic cycle (Sahoo et al., 2013 ). Therefore, the two SERCA-SLN complexes described above must alternately populate inhibitory interactions and secondary interactions that are non-inhibitory and release SERCA for progression through the transport cycle. This is most notable for the E2 SERCA-SLN complex that results in potent inhibition of SERCA. In the transition from the calcium-free E2 state to the calcium-bound E1 state, SLN must relocate away from the position transmembrane segment M2 will occupy. This requires a large change in how SLN interacts with SERCA. However, in the transition from the calcium-free E1-like state to the calcium-bound E1 state, side chain interactions between SLN and M2 are broken. This requires a small change in how SLN interacts with SERCA. Conformational memory refers to the exclusive nature of these SLN transitions during steady-state turnover of SERCA. In each case, SLN can reversibly transition between the initial inhibitory interaction (E2 or E1-like) and the associated secondary (non-inhibitory) interaction. These interactions are linked and SLN cannot transition to a different inhibitory interaction (from E2 to E1-like or E1-like to E2). Conformational memory suggests that the secondary, non-inhibitory interaction encodes information (memory) that allows SLN to return to the original inhibitory interaction with each new transport cycle (Fig. 7). The presence of conformational memory can be observed in the measurement of pre-steady-state charge translocation versus steady-state ATPase activity by SERCA (Fig. 1). In the charge translocation measurements, SERCA molecules undergo a single turnover event that loads the proteoliposomes with calcium and generates a current response (Tadini-Buoninsegni et al., 2006 ). Poising SERCA in different conformational states prior to initiating charge translocation influenced the kinetic parameters for SERCA alone, as well as SERCA in the presence of SLN. This was not surprising for the pre-steady-state measurements. In the ATPase activity measurements, SERCA molecules undergo continuous turnover under conditions where all variables and substrate concentrations remain constant. Poising SERCA in different conformational states prior to initiating ATPase activity influenced the kinetic parameters for both SERCA alone and SERCA in the presence of SLN, even under steady-state continuous turnover. This suggests that the initial inhibitory conformations of SERCA encode information on how the SERCA-SLN complex can progress through the calcium transport cycle, and this conformational memory is retained in multiple turnover events. MATERIALS & METHODS All reagents used were of the highest purity available. Reagents used for proteoliposome reconstitution include egg yolk phosphatidylcholine (PC), egg yolk phosphatidic acid (PA) (Avanti Polar Lipids, Alabaster AL), and octaethylene glycol monododecyl ether (C 12 E 8 ) (Barnet Products, Englewood Cliff, NJ). Reagents used for couple-enzyme ATPase measurements include ATP, NADH, phosphoenolpyruvate, pyruvate kinase, and lactate dehydrogenase (Sigma-Aldrich, Oakville, ON). Protein purification Recombinant human SLN was expressed and purified as previously described (Douglas et al., 2005 ; Gorski et al., 2013 ). SERCA1a was purified from rabbit skeletal muscle SR as described (Eletr & Inesi, 1972 ; Stokes & Green, 1990 ) with the following modifications. 100 mg of rabbit SR was suspended in extraction buffer (10 mg/mL C 12 E 8 , 8 mM CaCl 2 , 50 mM MOPS pH 7.0, 5 mM DTT, 20% glycerol) and stirred for 15 minutes at 4°C. Solubilized SR was centrifuged at 100,000 × g for 20 minutes and loaded onto a packed column containing Reactive Green Resin (Sigma-Aldrich, Oakville, ON) equilibrated with extraction buffer. The column was washed with two column volumes of wash buffer (1% C 12 E 8 , 1 mM CaCl 2 , 20 mM MOPS pH 7.0, 1 mM DTT, 20% glycerol) and eluted with elution buffer (10 mg/mL C 12 E 8 , 8 mM CaCl 2 , 50 mM MOPS pH 7.0, 5 mM DTT, 20% glycerol, 10 mM ADP, 50 mM NaCl). Elution fractions were evaluated for SERCA purity by SDS-PAGE and calcium-dependent ATPase activity. Co-reconstitution of SERCA and SLN Co-reconstitution of SLN and SERCA was performed as previously described (Glaves et al., 2020 ; Gorski et al., 2013 ) with the following modifications. 75 µg of lyophilized SLN was suspended in 100 µL trifluoroethanol: 2-propanol (5:1) and mixed with 360 µg egg yolk PC and 40 µg egg yolk PA. The mixture was then dried under N 2 (g) while vortexing to form a thin film of lipid and peptide, and placed under vacuum overnight. The lipid-peptide thin films were rehydrated in buffer (20 mM imidazole, pH 7.0, 100 mM KCl, and 0.02% NaN 3 ) at 37°C for 10 min, cooled to room temperature, and detergent solubilized by the addition of C 12 E 8 (0.2% final concentration) with vigorous vortexing. Detergent-solubilized SERCA1a was added (300 µg), and the reconstitution was stirred gently at room temperature. Detergent was slowly removed by the addition of Bio-Beads SM-2 resin (Bio-Rad Laboratories, Hercules, CA) over a 4-h time course (final ratio of 25 biobeads: 1 detergent w/w). After detergent removal, the reconstitution was centrifuged over a 20–50% sucrose step gradient for 1 h at 100,000 ×g. The resultant layer of reconstituted proteoliposomes was removed, flash frozen in liquid nitrogen and stored at -80°C. The final molar ratios were 120 lipids: 4.5 SLN: 1 SERCA and this SERCA-SLN ratio was confirmed by quantitative SDS-PAGE (Young, Jones & Stokes, 2001 ). Calcium-dependent ATPase activity measurements The calcium-dependent ATPase activity of SERCA1a in co-reconstituted proteoliposomes was determined by a coupled-enzyme assay over a range of calcium concentrations (0.1–10 µM). This assay has recently been adapted to a microplate reader and 96-well format (Armanious et al., 2024 ; Fisher et al., 2021 ). Proteoliposomes containing SERCA1a alone (negative control) and SERCA1a with SLN (experimental) were evaluated (~ 10–20 nM SERCA1a at 30°C). Reactions were initiated under different conditions that poised SERCA1a in distinct conformational states: (i) pre-incubation in the absence of substrates (substrate jump condition; 0.1–10 µM calcium, 4 mM ATP; assay initiated by the simultaneous addition of calcium and ATP); (ii) pre-incubation in the presence of calcium (ATP jump condition; 4 mM ATP; assay initiated by the addition of ATP); (iii) pre-incubation in the presence of ATP (calcium jump condition; 0.1–10 µM calcium; assay initiated by the addition of calcium); and (iv) physiological pre-incubation condition (physiological jump condition; 80 nM calcium, 4 mM ATP; assay initiated by the addition of calcium to achieve 0.1–10 µM calcium). Kinetic parameters V max (maximal activity), K Ca (apparent calcium affinity), and n H (cooperativity) were determined by nonlinear least-squares fitting of the activity data to the Hill equation (Sigma Plot software, SPSS, Chicago, IL). Errors were calculated as the standard error of the mean for a minimum of four independent reconstitutions. Comparison of the kinetic parameters was carried out using one-way analysis of variance (between subjects), followed by the Holm-Sidak test for pairwise comparisons. Charge translocation measurements Charge translocation measurements were performed with a SURFE 2 R N1 surface electrogenic event reader (Nanion Technologies, Munich, Germany). The temperature was maintained at ∼23°C. Charge movement was measured by adsorbing proteoliposomes containing SERCA in the absence and presence of SLN onto a solid supported membrane (Fig. 1; alkane thiol/phospholipid bilayer anchored to the surface of a gold electrode) (Smeazzetto et al., 2017 ; Tadini Buoninsegni et al., 2004 ). Following adsorption of the proteoliposomes to the SSM, charge translocation by SERCA was initiated by a concentration jump of a suitable substrate. The substrate pre-incubation and jump conditions (i-iv) are described above. These conditions induced charge translocation (calcium transport) across the proteoliposome membrane, and a current transient was recorded due to capacitive coupling between the proteoliposome and SSM ( Figure S1 ). The peak amplitude and numerically integrated current transient (total charge translocation) are related to the net charge movement in the proteoliposomes, which depends upon the electrogenic calcium transport by SERCA. The SSM technique detects pre-steady-state current transients within the first catalytic cycle of SERCA, and it is not sensitive to stationary currents following the first cycle (Tadini-Buoninsegni et al., 2006 ; Tadini-Buoninsegni et al., 2008 ). The peak amplitude (nA) and total charge translocation (nC) were plotted versus calcium concentration. Kinetic parameters V max (maximal activity), K Ca (apparent calcium affinity), and n H (cooperativity) were determined by nonlinear least-squares fitting of the activity data to the Hill equation (Sigma Plot software, SPSS, Chicago, IL). Errors were calculated as the standard error of the mean for a minimum of four independent reconstitutions. Comparison of the kinetic parameters was carried out using one-way analysis of variance (between subjects), followed by the Holm-Sidak test for pairwise comparisons. Declarations Data and Materials Availability All experimental data are available in the main text and supplemental data. COMPTING InterestS The authors declare that there are no competing interests associated with this manuscript. FUNDING This work was supported by grants from Heart and Stroke Foundation of Canada (HSY), the Canadian Institutes of Health Research (PJT-178282 to PL), and the Natural Sciences and Engineering Research Council of Canada (RGPIN-2016-06478 to MJL; RGPIN-04967 to PL). Author Contribution Conceptualization - JOP, MJL, HSY; Methodology - JOP, MJL, PL, HSY; Investigation - JOP, HSY; Visualization - JOP, HSY; Supervision – HSY; Writing – HSY; Writing, review & editing - JOP, HSY. Data Availability All experimental data are available in the main text and supplemental data. References Akin BL, Hurley TD, Chen Z, Jones LR (2013) The structural basis for phospholamban inhibition of the calcium pump in sarcoplasmic reticulum. J Biol Chem 288:30181–30191 Anderson DM, Anderson KM, Chang CL, Makarewich CA, Nelson BR, McAnally JR, Kasaragod P, Shelton JM, Liou J, Bassel-Duby R, Olson EN (2015) A micropeptide encoded by a putative long noncoding RNA regulates muscle performance. Cell 160:595–606 Anderson DM, Makarewich CA, Anderson KM, Shelton JM, Bezprozvannaya S, Bassel-Duby R, Olson EN (2016) Widespread control of calcium signaling by a family of SERCA-inhibiting micropeptides. Sci Signal 9:ra119 Armanious GP, Lemieux MJ, Espinoza-Fonseca LM, Young HS (2024) Missense variants in phospholamban and cardiac myosin binding protein identified in patients with a family history and clinical diagnosis of dilated cardiomyopathy. Biochim Biophys Acta Mol Cell Res 1871:119699 Autry JM, Thomas DD, Espinoza-Fonseca LM (2016) Sarcolipin Promotes Uncoupling of the SERCA Ca(2+) Pump by Inducing a Structural Rearrangement in the Energy-Transduction Domain. Biochemistry 55:6083–6086 Bal NC, Maurya SK, Sopariwala DH, Sahoo SK, Gupta SC, Shaikh SA, Pant M, Rowland LA, Bombardier E, Goonasekera SA, Tupling AR, Molkentin JD, Periasamy M (2012) Sarcolipin is a newly identified regulator of muscle-based thermogenesis in mammals. Nat Med 18:1575–1579 Ceholski DK, Trieber CA, Young HS (2012) Hydrophobic imbalance in the cytoplasmic domain of phospholamban is a determinant for lethal dilated cardiomyopathy. J Biol Chem 287:16521–16529 Csermely P, Kunsic N, Mendik P, Kerestely M, Farago T, Veres DV, Tompa P (2020) Learning of Signaling Networks: Molecular Mechanisms. Trends Biochem Sci 45:284–294 Douglas JL, Trieber CA, Afara M, Young HS (2005) Rapid, high-yield expression and purification of Ca2+-ATPase regulatory proteins for high-resolution structural studies. Protein Expr Purif 40:118–125 Eletr S, Inesi G (1972) Phospholipid orientation in sarcoplasmic membranes: spin-label ESR and proton MNR studies. Biochim Biophys Acta 282:174–179 Fisher ME, Bovo E, Aguayo-Ortiz R, Cho EE, Pribadi MP, Dalton MP, Rathod N, Lemieux MJ, Espinoza-Fonseca LM, Robia SL, Zima AV, Young HS (2021) Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA. Elife 10:e65545 Glaves JP, Primeau JO, Gorski PA, Espinoza-Fonseca LM, Lemieux MJ, Young HS (2020) Interaction of a Sarcolipin Pentamer and Monomer with the Sarcoplasmic Reticulum Calcium Pump, SERCA. Biophys J 118:518–531 Gorski PA, Glaves JP, Vangheluwe P, Young HS (2013) Sarco(endo)plasmic reticulum calcium ATPase (SERCA) inhibition by sarcolipin is encoded in its luminal tail. J Biol Chem 288:8456–8467 Gustavsson M, Verardi R, Mullen DG, Mote KR, Traaseth NJ, Gopinath T, Veglia G (2013) Allosteric regulation of SERCA by phosphorylation-mediated conformational shift of phospholamban. Proc Natl Acad Sci U S A 110:17338–17343 Maeda S, Taketsugu T, Morokuma K (2012) Automated Exploration of Photolytic Channels of HCOOH: Conformational Memory via Excited-State Roaming. J Phys Chem Lett 3:1900–1907 Makarov DE (2021) Barrier Crossing Dynamics from Single-Molecule Measurements. J Phys Chem B 125:2467–2476 Mall S, Broadbridge R, Harrison SL, Gore MG, Lee AG, East JM (2006) The presence of sarcolipin results in increased heat production by Ca(2+)-ATPase. J Biol Chem 281:36597–36602 Nelson BR, Makarewich CA, Anderson DM, Winders BR, Troupes CD, Wu F, Reese AL, McAnally JR, Chen X, Kavalali ET, Cannon SC, Houser SR, Bassel-Duby R, Olson EN (2016) A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle. Science 351:271–275 Odermatt A, Becker S, Khanna VK, Kurzydlowski K, Leisner E, Pette D, MacLennan DH (1998) Sarcolipin regulates the activity of SERCA1, the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase. J Biol Chem 273:12360–12369 Sahoo SK, Shaikh SA, Sopariwala DH, Bal NC, Bruhn DS, Kopec W, Khandelia H, Periasamy M (2015) The N Terminus of Sarcolipin Plays an Important Role in Uncoupling Sarco-endoplasmic Reticulum Ca2+-ATPase (SERCA) ATP Hydrolysis from Ca2 + Transport. J Biol Chem 290:14057–14067 Sahoo SK, Shaikh SA, Sopariwala DH, Bal NC, Periasamy M (2013) 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. J Biol Chem 288:6881–6889 Schorner M, Beyer SR, Southall J, Cogdell RJ, Kohler J (2015) Conformational Memory of a Protein Revealed by Single-Molecule Spectroscopy. J Phys Chem B 119:13964–13970 Seidel K, Andronesi OC, Krebs J, Griesinger C, Young HS, Becker S, Baldus M (2008) Structural characterization of Ca(2+)-ATPase-bound phospholamban in lipid bilayers by solid-state nuclear magnetic resonance (NMR) spectroscopy. Biochemistry 47:4369–4376 Smeazzetto S, Armanious GP, Moncelli MR, Bak JJ, Lemieux MJ, Young HS, Tadini-Buoninsegni F (2017) Conformational memory in the association of the transmembrane protein phospholamban with the sarcoplasmic reticulum calcium pump SERCA. J Biol Chem 292:21330–21339 Sorensen TL, Moller JV, Nissen P (2004) Phosphoryl transfer and calcium ion occlusion in the calcium pump. Science 304:1672–1675 Stokes DL, Green NM (1990) Three-dimensional crystals of CaATPase from sarcoplasmic reticulum. Symmetry and molecular packing. Biophys J 57:1–14 Tadini-Buoninsegni F (2020) Protein Adsorption on Solid Supported Membranes: Monitoring the Transport Activity of P-Type ATPases. Molecules 25 Tadini-Buoninsegni F, Bartolommei G, Moncelli MR, Guidelli R, Inesi G (2006) Pre-steady state electrogenic events of Ca2+/H+ exchange and transport by the Ca2+-ATPase. J Biol Chem 281:37720–37727 Tadini-Buoninsegni F, Bartolommei G, Moncelli MR, Tal DM, Lewis D, Inesi G (2008) Effects of high-affinity inhibitors on partial reactions, charge movements, and conformational States of the Ca2 + transport ATPase (sarco-endoplasmic reticulum Ca2 + ATPase). Mol Pharmacol 73:1134–1140 Tadini Buoninsegni F, Bartolommei G, Moncelli MR, Inesi G, Guidelli R (2004) Time-resolved charge translocation by sarcoplasmic reticulum Ca-ATPase measured on a solid supported membrane. Biophys J 86:3671–3686 Tian Y, Meng L, Zhang Z (2020) What is strain in neurodegenerative diseases? Cell Mol Life Sci 77:665–676 Toyoshima C, Asahi M, Sugita Y, Khanna R, Tsuda T, MacLennan DH (2003) Modeling of the inhibitory interaction of phospholamban with the Ca2 + ATPase. Proc Natl Acad Sci U S A 100:467–472 Toyoshima C, Iwasawa S, Ogawa H, Hirata A, Tsueda J, Inesi G (2013) Crystal structures of the calcium pump and sarcolipin in the Mg2+-bound E1 state. Nature 495:260–264 Toyoshima C, Mizutani T (2004) Crystal structure of the calcium pump with a bound ATP analogue. Nature 430:529–535 Toyoshima C, Nakasako M, Nomura H, Ogawa H (2000) Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution. Nature 405:647–655 Toyoshima C, Nomura H (2002) Structural changes in the calcium pump accompanying the dissociation of calcium. Nature 418:605–611 Trieber CA, Afara M, Young HS (2009) Effects of phospholamban transmembrane mutants on the calcium affinity, maximal activity, and cooperativity of the sarcoplasmic reticulum calcium pump. Biochemistry 48:9287–9296 Trieber CA, Douglas JL, Afara M, Young HS (2005) The effects of mutation on the regulatory properties of phospholamban in co-reconstituted membranes. Biochemistry 44:3289–3297 Tripathi T, Uversky VN, Giuliani A (2025) Intelligent' proteins. Cell Mol Life Sci 82:239 Winther AM, Bublitz M, Karlsen JL, Moller JV, Hansen JB, Nissen P, Buch-Pedersen MJ (2013) The sarcolipin-bound calcium pump stabilizes calcium sites exposed to the cytoplasm. Nature 495:265–269 Young HS, Jones LR, Stokes DL (2001) Locating phospholamban in co-crystals with Ca(2+)-ATPase by cryoelectron microscopy. Biophys J 81:884–894 Young HS, Rigaud JL, Lacapere JJ, Reddy LG, Stokes DL (1997) How to make tubular crystals by reconstitution of detergent-solubilized Ca2(+)-ATPase. Biophys J 72:2545–2558 Additional Declarations No competing interests reported. Supplementary Files floatimage8.png Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 08 May, 2026 Reviewers agreed at journal 30 Apr, 2026 Reviewers invited by journal 29 Apr, 2026 Editor assigned by journal 28 Apr, 2026 Submission checks completed at journal 28 Apr, 2026 First submitted to journal 27 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9544726","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":633327658,"identity":"28db7b04-e39f-48e2-9f59-82f562e7f651","order_by":0,"name":"Joseph O. 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10:57:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":675013,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9544726/v1/be7b0041ac8b94ae871eb04d.png"},{"id":108691434,"identity":"325c6f1e-d937-4cd7-929e-7aceb68f7e84","added_by":"auto","created_at":"2026-05-07 10:57:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":529301,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9544726/v1/fff21a4500dcec2b8109acd7.png"},{"id":108691448,"identity":"f3c587a7-adc2-463b-a051-217b2cecc74c","added_by":"auto","created_at":"2026-05-07 10:57:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":671988,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9544726/v1/6a5b3761babe170a4a01af57.png"},{"id":108691441,"identity":"3f855723-5508-4139-8a62-351f5b8d2962","added_by":"auto","created_at":"2026-05-07 10:57:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":714353,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9544726/v1/26432986139df22cb89a2640.png"},{"id":108691394,"identity":"dd47c070-a4d4-453e-9189-2d01927c7b59","added_by":"auto","created_at":"2026-05-07 10:57:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":488939,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9544726/v1/fbafd6b25bf810c47db7b695.png"},{"id":108691446,"identity":"a09ae63e-ee17-43c5-a360-7d21422a59aa","added_by":"auto","created_at":"2026-05-07 10:57:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1045020,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9544726/v1/203bea124965e0cb12620cf4.png"},{"id":108691469,"identity":"ac5a4aea-79bb-4f65-8a6a-0912722208a3","added_by":"auto","created_at":"2026-05-07 10:57:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":504415,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-9544726/v1/f8b20404314f5dd37dd5994b.png"},{"id":108805563,"identity":"22d277df-94df-4fb7-a93d-b808ea107311","added_by":"auto","created_at":"2026-05-08 15:26:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5063746,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9544726/v1/5e36e4db-b3d2-49b4-9fe8-1b47001541d3.pdf"},{"id":108691468,"identity":"a893f404-e78e-476b-8b5e-09e73470bc3d","added_by":"auto","created_at":"2026-05-07 10:57:48","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":711079,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-9544726/v1/612e34108e16f39bce1bab15.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sarcolipin is a conformation-dependent regulator of the sarcoplasmic reticulum calcium pump SERCA","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe sarco-endoplasmic reticulum calcium pump (SERCA) maintains the low resting-state calcium levels in the cytosol that are essential for many physiological signaling processes. A well-characterized example is the relaxation phase of the contraction-relaxation cycle in muscle. Following calcium release and muscle contraction, SERCA uses the energy of ATP to transport calcium ions into the sarcoplasmic reticulum (SR) lumen to achieve muscle relaxation. The calcium transport mechanism of SERCA has been extensively studied (e.g. (Sorensen, Moller \u0026amp; Nissen, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Toyoshima \u0026amp; Mizutani, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Toyoshima et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Toyoshima \u0026amp; Nomura, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2002\u003c/span\u003e)). SERCA progresses through a series of intermediates that alternate between high-affinity (E1) and low-affinity (E2) states for calcium. Calcium transport requires the binding of ATP (E2\u0026middot;ATP) and the cooperative binding of two calcium ions (E1\u0026middot;ATP\u0026middot;Ca) to form a catalytically competent conformation of SERCA. The energy of ATP is captured in the form of a phospho-enzyme intermediate (E1\u0026thinsp;~\u0026thinsp;P\u0026middot;Ca) and released through a conformational change that facilitates calcium translocation (E2-P). Following calcium release into the SR lumen, the phosphate is hydrolyzed and released, returning SERCA to a low-affinity state (E2). From this point, SERCA is poised to begin the transport cycle anew. It is important to note that the presence or absence of substrates can be used to poise SERCA in different conformational states (e.g. E2, E2\u0026middot;ATP, E1\u0026middot;Ca) and that the crystal structures of these conformational states have been determined.\u003c/p\u003e \u003cp\u003eSERCA activity is regulated by several tissue-specific transmembrane peptides called the regulins, which include phospholamban (PLN) and dwarf open reading frame (DWORF) in cardiac muscle and sarcolipin (SLN) and myoregulin (MLN) in skeletal muscle (Anderson et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Anderson et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Nelson et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). SLN is a 31-residue, α-helical integral membrane protein, consisting of a short cytoplasmic domain that contains a regulatory phosphorylation site, a transmembrane domain, and a highly conserved luminal tail. Like PLN, SLN has been reported to reduce the apparent calcium affinity of SERCA (Gorski et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Odermatt et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) by interacting with SERCA in an inhibitory groove formed by transmembrane helices M2, M6, and M9. Crystal structures revealed binding of SLN to the inhibitory groove of SERCA, which stabilized a calcium-free E1-like intermediate of SERCA that appears poised between the calcium-free E2 and calcium-bound E1 states (Toyoshima et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Winther et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The transmembrane portion of SLN mediates this interaction; however, SLN regulation is strongly dependent on the unique and highly conserved luminal Arg\u003csup\u003e27\u003c/sup\u003e-Ser-Tyr-Gln-Tyr\u003csup\u003e31\u003c/sup\u003e domain of SLN. While SLN was thought to be functionally homologous to PLN, the mechanism by which SLN regulates SERCA is distinct (Gorski et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to SLN\u0026rsquo;s role in regulating calcium homeostasis in skeletal muscle, it has been suggested that SLN also plays a role in thermogenesis and energy metabolism (Bal et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). SLN was reported to uncouple SERCA ATPase activity from calcium transport by promoting the backflow of calcium into the cytosol during ATP hydrolysis. A mechanism for SLN-mediated uncoupling through the interaction of SLN\u0026rsquo;s N-terminus with SERCA has been put forward (Autry, Thomas \u0026amp; Espinoza-Fonseca, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Given that skeletal muscle accounts for approximately 40% of adult body mass, the energy lost from SERCA uncoupling would make a significant contribution to heat generation and thermogenesis. This unique physiological role of SLN suggests a unique SERCA regulatory mechanism. The crystal structures of SERCA in the presence of SLN revealed a unexpected conformation of SERCA \u0026ndash; an E1-like, calcium-free state that appears poised between the E2 and E1 states (Toyoshima et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Winther et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). These structures are very similar to the crystal structure of SERCA-PLN complex (Akin et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). While these structures provide a plausible mechanism for SERCA inhibition, they yield little insight into the functional differences between PLN and SLN and the potential uncoupling behavior of SLN.\u003c/p\u003e \u003cp\u003eIn our previous work, we reported that the conformational state of SERCA influenced the interaction with PLN, suggesting that multiple conformational states of the SERCA-PLN complex were possible. Moreover, once a particular complex was engaged, the functional effect persisted through multiple SERCA turnover events of the catalytic cycle, and we termed this process \u0026ldquo;conformational memory\u0026rdquo; (Smeazzetto et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Herein, we performed a similar study of SERCA regulation by SLN. Through the use of particular substrates, SERCA was poised in the calcium-free E2 state (pre-incubation in the absence of substrates), the calcium-bound E1 state (pre-incubation with calcium), ATP-bound E2 state (pre-incubation with ATP), and a physiological resting state (pre-incubation with ATP \u0026amp; resting calcium). Proteoliposomes containing SERCA in the absence and presence of SLN were pre-incubated under these different substrate conditions. Following pre-incubation, calcium-dependent ATPase activity and charge translocation were measured. SLN decreased the maximal activity of SERCA under all substrate conditions tested. In contrast, SLN altered the apparent calcium affinity of SERCA only under one condition, pre-incubation in the absence of substrates. There was no evidence of SERCA uncoupling in the SERCA-SLN proteoliposomes. Nonetheless, the results indicated two possible SERCA-SLN regulatory complexes that depend on the conformation of SERCA. Once formed, these conformations are stable through steady-state turnover of SERCA. While both SERCA-PLN (Smeazzetto et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and SERCA-SLN complexes exhibited conformational memory, the results support the concept that SLN is a distinct regulatory subunit of SERCA.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe co-reconstituted proteoliposomes used in this study approximate the SR membrane in protein density, orientation, and lipid composition (Ceholski, Trieber \u0026amp; Young, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Gorski et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Trieber, Afara \u0026amp; Young, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Trieber et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Young et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). The proteoliposomes are unilamellar, SERCA molecules are oriented in the membrane with the cytoplasmic domains accessible to substrates on the outer surface (Young et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), and the lipid-to-protein ratio is ~\u0026thinsp;120 to 1 (Trieber et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). These proteoliposome characteristics mimic native skeletal muscle SR membranes, and they allow precise measurement of steady-state calcium-dependent ATPase activity (e.g. (Ceholski et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Gorski et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Trieber et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2009\u003c/span\u003e)) and pre-steady state calcium transport (Smeazzetto et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSERCA ATPase activity and calcium transport\u003c/h2\u003e \u003cp\u003eThe proteoliposomes containing SERCA in the absence and presence of SLN were pre-incubated under four different substrate conditions: (i) pre-incubation in the absence of substrates (substrate jump condition), (ii) pre-incubation with calcium (ATP jump condition), (iii) pre-incubation with ATP (calcium jump condition), and (iv) pre-incubation under physiological conditions (80 nM calcium, 4 mM ATP; physiological calcium jump condition). Following pre-incubation under these substrate conditions, we measured calcium-dependent ATP hydrolysis and calcium transport by SERCA (Fig.\u0026nbsp;1B, \u003cb\u003eC\u003c/b\u003e). ATP hydrolysis was measured as a function of calcium concentration (0.1\u0026ndash;10 \u0026micro;M free calcium). The data were fit to the Hill equation and the kinetic parameters maximal activity (V\u003csub\u003emax\u003c/sub\u003e) and apparent calcium affinity (K\u003csub\u003eCa\u003c/sub\u003e) were determined from the resultant curve fits (Fig.\u0026nbsp;2). The kinetic parameters obtained under various starting conditions are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCharge translocation by SERCA was also measured as a function of calcium concentration using a surface electrogenic even reader (SEER; SURFE\u003csup\u003e2\u003c/sup\u003eR N1, Nanion Technologies GmbH). Proteoliposomes containing SERCA in the absence and presence of SLN were adsorbed to a solid supported membrane (SSM) on the surface of a gold electrode as previously described (Smeazzetto et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Tadini-Buoninsegni, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) (Fig.\u0026nbsp;1C). The current transients observed following pre-incubation and initiation of the reaction were analyzed for peak current (nA) and total charge translocation (nC). The data were plotted as peak current (Fig.\u0026nbsp;3) and total charge translocation (Fig.\u0026nbsp;4) versus calcium concentration. These data were then fit to the Hill equation and the kinetic parameters maximal activity (V\u003csub\u003emax\u003c/sub\u003e) and apparent calcium affinity (K\u003csub\u003eCa\u003c/sub\u003e) were determined from the resultant curve fits (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Peak current measurements resulted in statistically significant differences in the maximal activity (V\u003csub\u003emax\u003c/sub\u003e) of SERCA, and charge translocation measurements resulted in statistically significant differences in the apparent calcium affinity (K\u003csub\u003eCa\u003c/sub\u003e) of SERCA. Overall, the trends observed with ATPase activity measurements (Fig.\u0026nbsp;2) were consistent with the charge translocation measurements (Figs.\u0026nbsp;3 \u0026amp; 4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKinetic parameters for SERCA and SERCA-SLN proteoliposomes derived from steady-state ATPase activity measurements and pre-steady state charge transfer measurements.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStarting conditions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eKinetic parameters\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eK\u003csub\u003eCa\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eV\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(\u0026micro;moles/min/mg)\u003c/p\u003e \u003cp\u003e(nA)\u003c/p\u003e \u003cp\u003e(nC)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003en\u003csub\u003eH\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eSubstrate jump\u003c/p\u003e \u003cp\u003ePre-incubate in the absence of substrates; start reaction with 4 mM ATP and 0.1 to 10 \u0026micro;M Ca\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA\u003csup\u003eATPase\u0026minus;activity\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003eATPase\u0026minus;activity\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA\u003csup\u003ePeak\u0026minus;current\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003ePeak\u0026minus;current\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA\u003csup\u003eCharge\u0026minus;translocation\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003eCharge\u0026minus;translocation\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eATP jump\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePre-incubate with 0.1 to 10 \u0026micro;M Ca\u003csup\u003e2+\u003c/sup\u003e; start reaction with 4 mM ATP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA\u003csup\u003eATPase\u0026minus;activity\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003eATPase\u0026minus;activity\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA\u003csup\u003ePeak\u0026minus;current\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003ePeak\u0026minus;current\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA\u003csup\u003eCharge\u0026minus;translocation\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003eCharge\u0026minus;translocation\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCalcium jump\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePre-incubate with 4 mM ATP; start reaction with 0.1 to 10 \u0026micro;M Ca\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA\u003csup\u003eATPase\u0026minus;activity\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003eATPase\u0026minus;activity\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA\u003csup\u003ePeak\u0026minus;current\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003ePeak\u0026minus;current\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA\u003csup\u003eCharge\u0026minus;translocation\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003eCharge\u0026minus;translocation\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePhysiological calcium jump\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePre-incubate with 4 mM ATP and 80 nM Ca\u003csup\u003e2+\u003c/sup\u003e; start reaction with remaining Ca\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA\u003csup\u003eATPase\u0026minus;activity\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003eATPase\u0026minus;activity\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA\u003csup\u003ePeak\u0026minus;current\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003ePeak\u0026minus;current\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA\u003csup\u003eCharge\u0026minus;translocation\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003eCharge\u0026minus;translocation\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues shaded in grey indicated SERCA and SERCA-SLN comparisons that were statistically significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.01).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSubstrate jump condition\u003c/h3\u003e\n\u003cp\u003eProteoliposomes containing SERCA in the absence and presence of SLN were pre-incubated in the absence of substrates. This pre-incubation condition favors the E2 state of SERCA, which is a low-affinity state for calcium binding. The substrate jump condition involved the simultaneous addition of calcium and ATP to initiate ATPase activity (Fig.\u0026nbsp;2 \u0026amp; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As previously observed (Gorski et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), SLN altered both the maximal activity (V\u003csub\u003emax\u003c/sub\u003e) and apparent calcium affinity (K\u003csub\u003eCa\u003c/sub\u003e) of SERCA. Proteoliposomes containing SERCA alone yielded a V\u003csub\u003emax\u003c/sub\u003e of 4.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u0026micro;mol/min/mg, while proteoliposomes containing SERCA and SLN yielded a V\u003csub\u003emax\u003c/sub\u003e of 2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u0026micro;mol/min/mg. Similarly, proteoliposomes containing SERCA alone yielded a K\u003csub\u003eCa\u003c/sub\u003e value of 0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;M calcium, compared to 0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u0026micro;M calcium for proteoliposomes containing SERCA and SLN. The increase in K\u003csub\u003eCa\u003c/sub\u003e indicates a decrease in the apparent calcium affinity of SERCA in the presence of SLN, which is a characteristic feature of SERCA inhibition. Thus, SLN decreased both the maximal activity and apparent calcium affinity of SERCA under these conditions.\u003c/p\u003e \u003cp\u003eThe substrate jump condition was next used to activate calcium transport by SERCA-containing proteoliposomes adsorbed to an SSM. Peak currents (nA) and total charge translocation (nC) were measured for current traces over a range of calcium concentrations. Proteoliposomes containing SERCA alone yielded a V\u003csub\u003emax\u003c/sub\u003e of 3.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 nA, while proteoliposomes containing SERCA and SLN yielded a V\u003csub\u003emax\u003c/sub\u003e of 2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 nA (Fig.\u0026nbsp;3). Similarly, the K\u003csub\u003eCa\u003c/sub\u003e values were found to be 0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 and 0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 \u0026micro;M calcium for SERCA in the absence and presence of SLN, respectively (Fig.\u0026nbsp;4). Thus, the V\u003csub\u003emax\u003c/sub\u003e and K\u003csub\u003eCa\u003c/sub\u003e values determined under these conditions using SEER technology were comparable to the values obtained from the calcium-dependent ATPase activity measurements (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eATP jump condition\u003c/h3\u003e\n\u003cp\u003eProteoliposomes containing SERCA in the absence and presence of SLN were next pre-incubated with calcium concentrations ranging from 0.1 to 10 \u0026micro;M before initiating SERCA ATPase activity (ATP jump; \u003cb\u003eFig.\u0026nbsp;2\u003c/b\u003e \u0026amp; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This pre-incubation condition favors the E1 state of SERCA, which is a high-affinity state for calcium binding. Compared to the substrate jump condition, the overall impact of SLN on SERCA activity was significantly reduced. The maximal activity of SERCA was lower with a modest depression in the presence of SLN (V\u003csub\u003emax\u003c/sub\u003e values of 3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u0026micro;mol/min/mg for SERCA alone compared to 2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u0026micro;mol/min/mg for SERCA-SLN). In addition, the effect of SLN on the apparent calcium affinity (K\u003csub\u003eCa\u003c/sub\u003e) of SERCA was more modest, and the observed K\u003csub\u003eCa\u003c/sub\u003e values were shifted to higher affinity (0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;M calcium for SERCA alone compared to 0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u0026micro;M calcium for SERCA-SLN). Consequently, pre-incubation of SERCA proteoliposomes with calcium resulted in a higher-affinity, slower pump, which manifested as an increase in the apparent calcium affinity of SERCA (decreased value for K\u003csub\u003eCa\u003c/sub\u003e) and a decrease in the maximal activity of SERCA. SLN inhibition of SERCA was still evident, but considerably diminished by pre-incubation with calcium.\u003c/p\u003e \u003cp\u003eThe ATP jump condition was used to activate calcium transport by SERCA-containing proteoliposomes adsorbed to an SSM using SEER technology. The measured charge translocation compared well with the ATPase activity measurements. Proteoliposomes containing SERCA alone yielded a V\u003csub\u003emax\u003c/sub\u003e of 4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 nA, while proteoliposomes containing SERCA and SLN yielded a V\u003csub\u003emax\u003c/sub\u003e of 3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 nA (Fig.\u0026nbsp;3). While there was an apparent reduction in V\u003csub\u003emax\u003c/sub\u003e, these values were not statistically different. K\u003csub\u003eCa\u003c/sub\u003e was found to be 0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 and 0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u0026micro;M calcium for SERCA in the absence and presence of SLN, respectively (Fig.\u0026nbsp;4). Thus, inhibition of SERCA calcium transport by SLN was also diminished following pre-incubation of SERCA in the presence of calcium (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eCalcium jump condition\u003c/h3\u003e\n\u003cp\u003eThe proteoliposomes containing SERCA in the absence and presence of SLN were pre-incubated with 4 mM ATP (absence of calcium), and SERCA ATPase activity was initiated by the addition of calcium (calcium jump condition; \u003cb\u003eFig.\u0026nbsp;2\u003c/b\u003e \u0026amp; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This pre-incubation condition favors the ATP-bound, calcium-free E2 state of SERCA. The V\u003csub\u003emax\u003c/sub\u003e values were determined to be 3.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u0026micro;mol/min/mg for SERCA alone compared to 2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u0026micro;mol/min/mg for SERCA in the presence of SLN. The V\u003csub\u003emax\u003c/sub\u003e values observed for pre-incubation with ATP were comparable to the substrate jump condition. In contrast, the effect of SLN on the apparent calcium affinity of SERCA was not observed with ATP pre-incubation (K\u003csub\u003eCa\u003c/sub\u003e of 0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;M calcium for SERCA alone compared to 0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u0026micro;M calcium for SERCA-SLN). These measurements indicated that ATP pre-incubation preserved the effect of SLN on the V\u003csub\u003emax\u003c/sub\u003e of SERCA. However, there was no change in the apparent calcium affinity of SERCA, indicating that this aspect of SLN inhibition was lost in the E2\u0026middot;ATP state of SERCA.\u003c/p\u003e \u003cp\u003eThe calcium jump condition was next used to activate calcium transport by SERCA-containing proteoliposomes. SERCA alone yielded a V\u003csub\u003emax\u003c/sub\u003e of 3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 nA, while proteoliposomes containing SERCA and SLN yielded a V\u003csub\u003emax\u003c/sub\u003e of 1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 nA (Fig.\u0026nbsp;3). This was the largest decrease in SERCA V\u003csub\u003emax\u003c/sub\u003e observed in the presence of SLN. K\u003csub\u003eCa\u003c/sub\u003e was found to be 0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 and 0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 \u0026micro;M calcium for SERCA in the absence and presence of SLN, respectively (Fig.\u0026nbsp;4). Thus, for the ATPase activity and charge translocation measurements, the primary effect of SLN on SERCA under ATP pre-incubation conditions was depression of SERCA\u0026rsquo;s V\u003csub\u003emax\u003c/sub\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003ePhysiological calcium jump\u003c/h3\u003e\n\u003cp\u003eThe different pre-incubation conditions described above poised SERCA in well-known conformational states \u0026ndash; E2, E1, and E2\u0026middot;ATP, respectively. However, these pre-incubation conditions differ from the physiological resting state (mM ATP and nM calcium). To mimic the physiological resting state, SERCA and SERCA-SLN proteoliposomes were pre-incubated with 4 mM ATP and 80 nM calcium, near the resting level of cytosolic calcium that myocytes experience. The ATPase activity of SERCA and SERCA-SLN proteoliposomes under these conditions was determined (physiological calcium jump condition; \u003cb\u003eFig.\u0026nbsp;2\u003c/b\u003e \u0026amp; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The observed V\u003csub\u003emax\u003c/sub\u003e depression remained between SERCA and SERCA-SLN proteoliposomes, though it was reduced (V\u003csub\u003emax\u003c/sub\u003e values of 3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 and 3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u0026micro;mol/min/mg, respectively). The K\u003csub\u003eCa\u003c/sub\u003e shift observed between SERCA and SERCA-SLN proteoliposomes was also reduced (K\u003csub\u003eCa\u003c/sub\u003e values of 0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 and 0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u0026micro;M calcium, respectively). This small change in the apparent calcium affinity of SERCA was akin to the weak SLN inhibition observed for the calcium pre-incubation (ATP jump) and ATP pre-incubation (calcium jump) conditions. These trends in the presence of SLN \u0026ndash; V\u003csub\u003emax\u003c/sub\u003e depression and weak SERCA inhibition \u0026ndash; agreed with previous observations for SERCA in the presence of PLN (Smeazzetto et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe physiological calcium jump condition was next used to activate calcium transport by SERCA-containing proteoliposomes. Similar to the ATPase activity measurements for this pre-incubation condition, the effects of SLN on the V\u003csub\u003emax\u003c/sub\u003e (3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 and 2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 nA respectively; \u003cb\u003eFig.\u0026nbsp;3\u003c/b\u003e) and K\u003csub\u003eCa\u003c/sub\u003e (0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 and 0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u0026micro;M calcium, respectively; \u003cb\u003eFig.\u0026nbsp;4\u003c/b\u003e) of SERCA were reduced. Thus, the primary effect of SLN on SERCA under physiological pre-incubation conditions was depression of SERCA\u0026rsquo;s V\u003csub\u003emax\u003c/sub\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These data are consistent with the ATPase activity of SERCA in the absence and presence of SLN (Fig.\u0026nbsp;2 \u0026amp; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSummary of Results\u003c/h2\u003e \u003cp\u003eSLN consistently decreased the maximal activity (V\u003csub\u003emax\u003c/sub\u003e) of SERCA under all conditions tested, using both ATPase activity and charge translocation as measures of SERCA function. This effect was more pronounced under calcium-free pre-incubation conditions that promoted the E2 state of SERCA (Figs.\u0026nbsp;2 \u0026amp; 3; substrate jump \u0026amp; calcium jump conditions) and diminished under calcium pre-incubation conditions that promoted the E1 state of SERCA (Figs.\u0026nbsp;2 \u0026amp; 4; ATP jump \u0026amp; physiological calcium jump conditions). SLN altered the apparent calcium affinity of SERCA (K\u003csub\u003eCa\u003c/sub\u003e) under one condition (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; substrate jump condition), though there were minor changes in K\u003csub\u003eCa\u003c/sub\u003e under most other conditions.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eComparative charge translocation by SERCA, SERCA-SLN, and SERCA-PLN\u003c/h3\u003e\n\u003cp\u003eSLN has been reported to uncouple SERCA calcium transport from ATP hydrolysis as a means of heat production in non-shivering thermogenesis (Bal et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Mall et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Sahoo et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This is considered a unique property of SLN regulation of SERCA in skeletal muscle, and not a property of PLN regulation of SERCA in cardiac muscle. To investigate the possible uncoupling of calcium transport from ATP hydrolysis, we compared the ATP hydrolysis rates (V\u003csub\u003emax\u003c/sub\u003e in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) with the total charge translocation for SERCA in the absence and presence of SLN (\u003cb\u003eFigure S1\u003c/b\u003e). The underlying assumption was that uncoupling should manifest as a selective reduction in charge movement for SERCA in the presence of SLN. We also compared the total charge translocation by SERCA in the presence of SLN to SERCA in the presence of PLN (Fig.\u0026nbsp;5). No differences were observed in the kinetic parameters derived from total charge translocation (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), suggesting that calcium transport is similar in SERCA-SLN and SERCA-PLN proteoliposomes. In addition, under most pre-incubation conditions, the maximal activity of SERCA in ATPase activity measurements (V\u003csub\u003emax\u003c/sub\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) was higher for SERCA-PLN compared to SERCA-SLN. This suggests that coupling of ATP-hydrolysis to charge-translocation was higher for SERCA-PLN and lower for SERCA-SLN. Since we would expect a signature feature of SERCA uncoupling to involve higher ATP hydrolysis and lower calcium transport, this is the opposite trend expected for uncoupling of SERCA by SLN.\u003c/p\u003e \u003cp\u003eWhile there was no evidence of SERCA uncoupling in the SERCA-SLN proteoliposomes, there were advantages and disadvantages in using this membrane reconstitution system. The SERCA-SLN proteoliposomes allowed detailed functional measurements and mechanistic insights in a highly purified system, though they lacked the complexity of the cell and additional factors that may modulate cellular calcium homeostasis. The combination of calcium-dependent ATPase activity and charge translocation measurements allowed comparison of both ATP hydrolysis and calcium transport by SERCA. However, the charge translocation measurements were not quantitative in terms of the number of transported calcium ions or the number of SERCA molecules contributing to the signal. To overcome this, we compared the relative charge translocation by SERCA, SERCA-SLN, and SERCA-PLN proteoliposomes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKinetic parameters for SERCA-SLN and SERCA-PLN proteoliposomes derived from steady-state ATPase activity measurements and pre-steady state charge transfer measurements (total charge translocation).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStarting conditions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eKinetic parameters\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eK\u003csub\u003eCa\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eV\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(nC)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003en\u003csub\u003eH\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eSubstrate jump\u003c/p\u003e \u003cp\u003ePre-incubate in the absence of substrates; start reaction with 4 mM ATP and 0.1 to 10 \u0026micro;M Ca\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003eATPase\u0026minus;activity\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-PLN\u003csup\u003eATPase\u0026minus;activity\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003eCharge\u0026minus;translocation\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-PLN\u003csup\u003eCharge\u0026minus;translocation\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eATP jump\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePre-incubate with 0.1 to 10 \u0026micro;M Ca\u003csup\u003e2+\u003c/sup\u003e; start reaction with 4 mM ATP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003eATPase\u0026minus;activity\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-PLN\u003csup\u003eATPase\u0026minus;activity\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003eCharge\u0026minus;translocation\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-PLN\u003csup\u003eCharge\u0026minus;translocation\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCalcium jump\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePre-incubate with 4 mM ATP; start reaction with 0.1 to 10 \u0026micro;M Ca\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003eATPase\u0026minus;activity\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-PLN\u003csup\u003eATPase\u0026minus;activity\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003eCharge\u0026minus;translocation\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-PLN\u003csup\u003eCharge\u0026minus;translocation\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePhysiological calcium jump\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePre-incubate with 4 mM ATP and 80 nM Ca\u003csup\u003e2+\u003c/sup\u003e; start reaction with remaining Ca\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003eATPase\u0026minus;activity\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-PLN\u003csup\u003eATPase\u0026minus;activity\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-SLN\u003csup\u003eCharge\u0026minus;translocation\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSERCA-PLN\u003csup\u003eCharge\u0026minus;translocation\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe pre-incubation conditions described above poised SERCA in two main conformational states: calcium-free E2 and calcium-bound E1. The calcium-free E2 state was promoted by the substrate jump condition (pre-incubation in the absence of substrates) and the calcium jump condition (pre-incubation in the presence of ATP). The calcium-bound E1 state was promoted by the ATP jump condition (pre-incubation in the presence of calcium) and the physiological calcium jump condition (pre-incubation in resting-state conditions). The E2 substrate jump condition is our typical method for initiating functional measurements of SERCA in the presence of SLN (Gorski et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Under this condition, SERCA and SLN are pre-incubated in the absence of calcium and ATP, and the simultaneous addition of substrates initiated SERCA ATPase activity and calcium transport. As a result, the inhibition of SERCA by SLN manifested as a decrease in both the apparent calcium affinity and maximal activity of SERCA (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The decrease in the apparent calcium affinity of SERCA in the presence of SLN is consistent with previous observations by others (Odermatt et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1998\u003c/span\u003e); however, this effect of SLN was only observed for the substrate jump condition. The primary effect of SLN under all conditions was a depression of SERCA\u0026rsquo;s maximal activity. This effect was more pronounced for the E2 states of SERCA and diminished for the E1 states of SERCA. Thus, the interaction of SLN with SERCA exhibited a clear preference for the calcium-free E2 states of SERCA.\u003c/p\u003e \u003cp\u003eThe physiological jump condition pre-incubated SERCA under resting-state physiological conditions followed by a calcium jump to elicit SERCA ATPase activity and charge translocation. Under this pre-incubation condition, SERCA slowly progresses through the calcium transport cycle. The turnover number was approximately 11 per minute (0.1 \u0026micro;moles/min/mg at 25˚C), while SERCA\u0026rsquo;s maximum turnover rate in proteoliposomes was ~\u0026thinsp;450 per minute for SERCA alone (4.1 \u0026micro;moles/min/mg at 25˚C). The slow turnover of SERCA through the calcium transport cycle presumably allowed SLN to interact with any preferred conformational state. That said, SERCA may preferentially occupy states that precede the rate-limiting steps in the reaction cycle, particularly the E1\u0026thinsp;~\u0026thinsp;P to E2P transition. Thus, the E1\u0026thinsp;~\u0026thinsp;P conformation of SERCA was a likely candidate for SLN interaction. Consistent with this notion, SERCA regulation by SLN was most similar to the calcium pre-incubation condition (ATP jump; Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We categorized this as a condition that promoted the calcium-bound E1 state of SERCA, which could explain the diminished effect of SLN on SERCA activity (Fig.\u0026nbsp;2, 3, \u0026amp;, \u003cb\u003e4\u003c/b\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSERCA apparent calcium affinity and maximal activity\u003c/h2\u003e \u003cp\u003eUnder most conditions tested, only a slight shift in the apparent calcium affinity of SERCA was observed in the presence of SLN (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Surprisingly, the presence of substrates \u0026ndash; calcium, ATP, or both \u0026ndash; resulted in a weak effect of SLN on the apparent calcium affinity of SERCA. The exception was pre-incubation of SERCA in the absence of substrates, which resulted in a more inhibitory interaction of SLN and a significant shift in the apparent calcium affinity of SERCA (substrate jump condition; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results suggested that the physical interaction between SERCA and SLN was sensitive to the conformational state of SERCA and that at least two distinct SERCA-SLN complexes exist. One complex caused a decrease in the maximal activity of SERCA. This was most pronounced for the complex of SLN with the calcium-free E2 states of SERCA, but clearly present under all SERCA conformations tested. A second complex resulted in a decrease in both the apparent calcium affinity and maximal activity of SERCA. These results were unique for the complex of SLN with the substrate-free conformation of SERCA (E2). Surprisingly, this was not observed for the complex of SLN with the calcium-free, ATP-bound conformation (E2\u0026middot;ATP) of SERCA. In a previous study of the SERCA-PLN complex (Smeazzetto et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), we reached a similar conclusion that the calcium-free E2\u0026middot;ATP state of SERCA had distinct structural and functional properties from the substrate-free E2 state of SERCA. This was a surprising finding given that the structures of SERCA in the E2 and E2\u0026middot;ATP states are similar (e.g. compare PDB 1IWO with 2C88 or 3FGO, respectively).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSERCA-SLN complexes\u003c/h2\u003e \u003cp\u003eThe results described above point toward two distinct conformational states for the SERCA-SLN complex. One conformation resulted in weaker inhibition of SERCA with minor changes in SERCA kinetics (pre-incubation with calcium in the ATP jump \u0026amp; physiological jump conditions). A second conformation resulted in potent inhibition of SERCA with large changes in SERCA kinetics (pre-incubation without calcium in the substrate jump \u0026amp; calcium jump conditions). We suggest that the weaker inhibitory state corresponds to the known crystal structure of the SERCA-SLN complex (Toyoshima et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Winther et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and the potent inhibitory state corresponds to the previous model of the SERCA-PLN complex (Seidel et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Toyoshima et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOne conformation must correspond to the crystal structure of the SERCA-SLN complex (Toyoshima et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Winther et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), where SERCA is found in a calcium-free E1-like state (Fig.\u0026nbsp;6A,\u003cb\u003eB\u003c/b\u003e). This calcium-free E1-like state is closer to the calcium-bound E1 state of SERCA than it is to the calcium-free E2 state. Nonetheless, SLN interacts with M2, M6, and M9 of SERCA to form an inhibitory complex. The nature of the complex suggests that SLN slows transmembrane domain movements that allow SERCA to progress through the calcium transport cycle. However, as SERCA progresses through the transport cycle, SLN must alternately populate two interaction sites \u0026ndash; one site that is inhibitory and a second site that is non-inhibitory and releases SERCA for progression through the transport cycle. In the transition from the calcium-free E1-like state to the calcium-bound E1 state of SERCA, the upward movement of transmembrane segment M2 is a major structural change. Side-chain interactions mediate the SLN complex with M2, and only a small change in the position of SLN is required to disengage. Since this complex is poised closer to the calcium-bound state of SERCA, it appears consistent with the weaker inhibitory effect of SLN under some SERCA pre-incubation conditions (ATP jump and physiological jump conditions, which pre-incubated SERCA in the presence of calcium).\u003c/p\u003e \u003cp\u003eWe suggest that the second conformation corresponds to the calcium-free E2 state of SERCA, as predicted by the original molecular models for the SERCA-PLN complex (Gustavsson et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Seidel et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Toyoshima et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) (Fig.\u0026nbsp;6C,\u003cb\u003eD\u003c/b\u003e). The two conformations, the E1-like state and the E2 state, differ in how SLN is oriented in the inhibitory groove of SERCA formed by transmembrane segments M2, M6, and M9 (Fig.\u0026nbsp;6E,\u003cb\u003eF \u0026amp; inset\u003c/b\u003e). The binding groove is shallow (partially closed) in the E1-like state of SERCA and deeper (fully open) in the E2 state. This allows SLN to penetrate further into the inhibitory groove in E2, which results in a 5\u0026ndash;7 \u0026Aring; lateral translation of SLN compared to the E1-like complex. The position of SLN in the E2 complex partially occupies the position M2 will take in the transition to the calcium-bound E1 state of SERCA. While SLN needs to relocate to allow the movement of M2, the repositioning of SLN required in the E2 complex is larger compared to the E1-like complex. Since this complex is poised further from the calcium-bound state of SERCA and SLN partially occupies the position M2 will take, it appears consistent with the potent inhibitory effect of SLN under some SERCA pre-incubation conditions. These include the substrate jump and calcium jump conditions, which pre-incubated SERCA in the absence of calcium and promoted the calcium-free E2 state. Thus, these conditions seemed consistent with an E2 SERCA-SLN complex that has a potent inhibitory effect on SERCA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eConformational memory\u003c/h2\u003e \u003cp\u003eThe concept of conformational memory has been explored in protein conformational dynamics, inherently disordered proteins, amyloids, and chemical reactions (Csermely et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Maeda, Taketsugu \u0026amp; Morokuma, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Makarov, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Schorner et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Tian, Meng \u0026amp; Zhang, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Several proteins exhibit conformational memory by retaining information about past conformations or interactions that influence future activity, e.g. the retention of a ligand bound state after ligand dissociation (Csermely et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tripathi, Uversky \u0026amp; Giuliani, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). SERCA is a molecular machine that undergoes a wide range of conformational states during the transport cycle, all of which require dynamics and flexibility in domain motions. The transitions between these different conformational states of SERCA have been assumed to include spontaneous conformational fluctuations in a stochastic process. However, the availability of substrates can influence the energy landscape for conformational fluctuations and shift from a stochastic reaction coordinate to one with reduced degrees of freedom between SERCA intermediates (conformational memory). The same argument can be made for the interaction of SLN with SERCA. The reaction coordinate between SERCA inhibition by SLN and SERCA\u0026rsquo;s progression through the calcium transport cycle is not stochastic. We suggest that SLN cycles between inhibitory and non-inhibitory interactions with SERCA, and that these interactions are linked by conformational memory \u0026ndash; past SERCA-SLN interactions influence future transport cycles (Fig.\u0026nbsp;7).\u003c/p\u003e \u003cp\u003eIt is important to remember that SLN remains associated with SERCA during the catalytic cycle (Sahoo et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Therefore, the two SERCA-SLN complexes described above must alternately populate inhibitory interactions and secondary interactions that are non-inhibitory and release SERCA for progression through the transport cycle. This is most notable for the E2 SERCA-SLN complex that results in potent inhibition of SERCA. In the transition from the calcium-free E2 state to the calcium-bound E1 state, SLN must relocate away from the position transmembrane segment M2 will occupy. This requires a large change in how SLN interacts with SERCA. However, in the transition from the calcium-free E1-like state to the calcium-bound E1 state, side chain interactions between SLN and M2 are broken. This requires a small change in how SLN interacts with SERCA. Conformational memory refers to the exclusive nature of these SLN transitions during steady-state turnover of SERCA. In each case, SLN can reversibly transition between the initial inhibitory interaction (E2 or E1-like) and the associated secondary (non-inhibitory) interaction. These interactions are linked and SLN cannot transition to a different inhibitory interaction (from E2 to E1-like or E1-like to E2). Conformational memory suggests that the secondary, non-inhibitory interaction encodes information (memory) that allows SLN to return to the original inhibitory interaction with each new transport cycle (Fig.\u0026nbsp;7).\u003c/p\u003e \u003cp\u003eThe presence of conformational memory can be observed in the measurement of pre-steady-state charge translocation versus steady-state ATPase activity by SERCA (Fig.\u0026nbsp;1). In the charge translocation measurements, SERCA molecules undergo a single turnover event that loads the proteoliposomes with calcium and generates a current response (Tadini-Buoninsegni et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Poising SERCA in different conformational states prior to initiating charge translocation influenced the kinetic parameters for SERCA alone, as well as SERCA in the presence of SLN. This was not surprising for the pre-steady-state measurements. In the ATPase activity measurements, SERCA molecules undergo continuous turnover under conditions where all variables and substrate concentrations remain constant. Poising SERCA in different conformational states prior to initiating ATPase activity influenced the kinetic parameters for both SERCA alone and SERCA in the presence of SLN, even under steady-state continuous turnover. This suggests that the initial inhibitory conformations of SERCA encode information on how the SERCA-SLN complex can progress through the calcium transport cycle, and this conformational memory is retained in multiple turnover events.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e "},{"header":"MATERIALS \u0026 METHODS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003cp\u003eAll reagents used were of the highest purity available. Reagents used for proteoliposome reconstitution include egg yolk phosphatidylcholine (PC), egg yolk phosphatidic acid (PA) (Avanti Polar Lipids, Alabaster AL), and octaethylene glycol monododecyl ether (C\u003csub\u003e12\u003c/sub\u003eE\u003csub\u003e8\u003c/sub\u003e) (Barnet Products, Englewood Cliff, NJ). Reagents used for couple-enzyme ATPase measurements include ATP, NADH, phosphoenolpyruvate, pyruvate kinase, and lactate dehydrogenase (Sigma-Aldrich, Oakville, ON).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eProtein purification\u003c/h2\u003e \u003cp\u003eRecombinant human SLN was expressed and purified as previously described (Douglas et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Gorski et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). SERCA1a was purified from rabbit skeletal muscle SR as described (Eletr \u0026amp; Inesi, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1972\u003c/span\u003e; Stokes \u0026amp; Green, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) with the following modifications. 100 mg of rabbit SR was suspended in extraction buffer (10 mg/mL C\u003csub\u003e12\u003c/sub\u003eE\u003csub\u003e8\u003c/sub\u003e, 8 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 50 mM MOPS pH 7.0, 5 mM DTT, 20% glycerol) and stirred for 15 minutes at 4\u0026deg;C. Solubilized SR was centrifuged at 100,000 \u0026times; g for 20 minutes and loaded onto a packed column containing Reactive Green Resin (Sigma-Aldrich, Oakville, ON) equilibrated with extraction buffer. The column was washed with two column volumes of wash buffer (1% C\u003csub\u003e12\u003c/sub\u003eE\u003csub\u003e8\u003c/sub\u003e, 1 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 20 mM MOPS pH 7.0, 1 mM DTT, 20% glycerol) and eluted with elution buffer (10 mg/mL C\u003csub\u003e12\u003c/sub\u003eE\u003csub\u003e8\u003c/sub\u003e, 8 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 50 mM MOPS pH 7.0, 5 mM DTT, 20% glycerol, 10 mM ADP, 50 mM NaCl). Elution fractions were evaluated for SERCA purity by SDS-PAGE and calcium-dependent ATPase activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCo-reconstitution of SERCA and SLN\u003c/h2\u003e \u003cp\u003eCo-reconstitution of SLN and SERCA was performed as previously described (Glaves et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gorski et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) with the following modifications. 75 \u0026micro;g of lyophilized SLN was suspended in 100 \u0026micro;L trifluoroethanol: 2-propanol (5:1) and mixed with 360 \u0026micro;g egg yolk PC and 40 \u0026micro;g egg yolk PA. The mixture was then dried under N\u003csub\u003e2\u003c/sub\u003e(g) while vortexing to form a thin film of lipid and peptide, and placed under vacuum overnight. The lipid-peptide thin films were rehydrated in buffer (20 mM imidazole, pH 7.0, 100 mM KCl, and 0.02% NaN\u003csub\u003e3\u003c/sub\u003e) at 37\u0026deg;C for 10 min, cooled to room temperature, and detergent solubilized by the addition of C\u003csub\u003e12\u003c/sub\u003eE\u003csub\u003e8\u003c/sub\u003e (0.2% final concentration) with vigorous vortexing. Detergent-solubilized SERCA1a was added (300 \u0026micro;g), and the reconstitution was stirred gently at room temperature. Detergent was slowly removed by the addition of Bio-Beads SM-2 resin (Bio-Rad Laboratories, Hercules, CA) over a 4-h time course (final ratio of 25 biobeads: 1 detergent w/w). After detergent removal, the reconstitution was centrifuged over a 20\u0026ndash;50% sucrose step gradient for 1 h at 100,000 \u0026times;g. The resultant layer of reconstituted proteoliposomes was removed, flash frozen in liquid nitrogen and stored at -80\u0026deg;C. The final molar ratios were 120 lipids: 4.5 SLN: 1 SERCA and this SERCA-SLN ratio was confirmed by quantitative SDS-PAGE (Young, Jones \u0026amp; Stokes, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCalcium-dependent ATPase activity measurements\u003c/h2\u003e \u003cp\u003eThe calcium-dependent ATPase activity of SERCA1a in co-reconstituted proteoliposomes was determined by a coupled-enzyme assay over a range of calcium concentrations (0.1\u0026ndash;10 \u0026micro;M). This assay has recently been adapted to a microplate reader and 96-well format (Armanious et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Fisher et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Proteoliposomes containing SERCA1a alone (negative control) and SERCA1a with SLN (experimental) were evaluated (~\u0026thinsp;10\u0026ndash;20 nM SERCA1a at 30\u0026deg;C). Reactions were initiated under different conditions that poised SERCA1a in distinct conformational states: (i) pre-incubation in the absence of substrates (substrate jump condition; 0.1\u0026ndash;10 \u0026micro;M calcium, 4 mM ATP; assay initiated by the simultaneous addition of calcium and ATP); (ii) pre-incubation in the presence of calcium (ATP jump condition; 4 mM ATP; assay initiated by the addition of ATP); (iii) pre-incubation in the presence of ATP (calcium jump condition; 0.1\u0026ndash;10 \u0026micro;M calcium; assay initiated by the addition of calcium); and (iv) physiological pre-incubation condition (physiological jump condition; 80 nM calcium, 4 mM ATP; assay initiated by the addition of calcium to achieve 0.1\u0026ndash;10 \u0026micro;M calcium). Kinetic parameters V\u003csub\u003emax\u003c/sub\u003e (maximal activity), K\u003csub\u003eCa\u003c/sub\u003e (apparent calcium affinity), and n\u003csub\u003eH\u003c/sub\u003e (cooperativity) were determined by nonlinear least-squares fitting of the activity data to the Hill equation (Sigma Plot software, SPSS, Chicago, IL). Errors were calculated as the standard error of the mean for a minimum of four independent reconstitutions. Comparison of the kinetic parameters was carried out using one-way analysis of variance (between subjects), followed by the Holm-Sidak test for pairwise comparisons.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCharge translocation measurements\u003c/h2\u003e \u003cp\u003eCharge translocation measurements were performed with a SURFE\u003csup\u003e2\u003c/sup\u003eR N1 surface electrogenic event reader (Nanion Technologies, Munich, Germany). The temperature was maintained at \u0026sim;23\u0026deg;C. Charge movement was measured by adsorbing proteoliposomes containing SERCA in the absence and presence of SLN onto a solid supported membrane (Fig.\u0026nbsp;1; alkane thiol/phospholipid bilayer anchored to the surface of a gold electrode) (Smeazzetto et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Tadini Buoninsegni et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Following adsorption of the proteoliposomes to the SSM, charge translocation by SERCA was initiated by a concentration jump of a suitable substrate. The substrate pre-incubation and jump conditions (i-iv) are described above. These conditions induced charge translocation (calcium transport) across the proteoliposome membrane, and a current transient was recorded due to capacitive coupling between the proteoliposome and SSM (\u003cb\u003eFigure S1\u003c/b\u003e). The peak amplitude and numerically integrated current transient (total charge translocation) are related to the net charge movement in the proteoliposomes, which depends upon the electrogenic calcium transport by SERCA. The SSM technique detects pre-steady-state current transients within the first catalytic cycle of SERCA, and it is not sensitive to stationary currents following the first cycle (Tadini-Buoninsegni et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Tadini-Buoninsegni et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The peak amplitude (nA) and total charge translocation (nC) were plotted versus calcium concentration. Kinetic parameters V\u003csub\u003emax\u003c/sub\u003e (maximal activity), K\u003csub\u003eCa\u003c/sub\u003e (apparent calcium affinity), and n\u003csub\u003eH\u003c/sub\u003e (cooperativity) were determined by nonlinear least-squares fitting of the activity data to the Hill equation (Sigma Plot software, SPSS, Chicago, IL). Errors were calculated as the standard error of the mean for a minimum of four independent reconstitutions. Comparison of the kinetic parameters was carried out using one-way analysis of variance (between subjects), followed by the Holm-Sidak test for pairwise comparisons.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eData and Materials Availability\u003c/h2\u003e \u003cp\u003eAll experimental data are available in the main text and supplemental data.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCOMPTING InterestS\u003c/strong\u003e \u003cp\u003eThe authors declare that there are no competing interests associated with this manuscript.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eThis work was supported by grants from Heart and Stroke Foundation of Canada (HSY), the Canadian Institutes of Health Research (PJT-178282 to PL), and the Natural Sciences and Engineering Research Council of Canada (RGPIN-2016-06478 to MJL; RGPIN-04967 to PL).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization - JOP, MJL, HSY; Methodology - JOP, MJL, PL, HSY; Investigation - JOP, HSY; Visualization - JOP, HSY; Supervision \u0026ndash; HSY; Writing \u0026ndash; HSY; Writing, review \u0026amp; editing - JOP, HSY.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll experimental data are available in the main text and supplemental data.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAkin BL, Hurley TD, Chen Z, Jones LR (2013) The structural basis for phospholamban inhibition of the calcium pump in sarcoplasmic reticulum. J Biol Chem 288:30181\u0026ndash;30191\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnderson DM, Anderson KM, Chang CL, Makarewich CA, Nelson BR, McAnally JR, Kasaragod P, Shelton JM, Liou J, Bassel-Duby R, Olson EN (2015) A micropeptide encoded by a putative long noncoding RNA regulates muscle performance. Cell 160:595\u0026ndash;606\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnderson DM, Makarewich CA, Anderson KM, Shelton JM, Bezprozvannaya S, Bassel-Duby R, Olson EN (2016) Widespread control of calcium signaling by a family of SERCA-inhibiting micropeptides. Sci Signal 9:ra119\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArmanious GP, Lemieux MJ, Espinoza-Fonseca LM, Young HS (2024) Missense variants in phospholamban and cardiac myosin binding protein identified in patients with a family history and clinical diagnosis of dilated cardiomyopathy. Biochim Biophys Acta Mol Cell Res 1871:119699\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAutry JM, Thomas DD, Espinoza-Fonseca LM (2016) Sarcolipin Promotes Uncoupling of the SERCA Ca(2+) Pump by Inducing a Structural Rearrangement in the Energy-Transduction Domain. Biochemistry 55:6083\u0026ndash;6086\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBal NC, Maurya SK, Sopariwala DH, Sahoo SK, Gupta SC, Shaikh SA, Pant M, Rowland LA, Bombardier E, Goonasekera SA, Tupling AR, Molkentin JD, Periasamy M (2012) Sarcolipin is a newly identified regulator of muscle-based thermogenesis in mammals. Nat Med 18:1575\u0026ndash;1579\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCeholski DK, Trieber CA, Young HS (2012) Hydrophobic imbalance in the cytoplasmic domain of phospholamban is a determinant for lethal dilated cardiomyopathy. J Biol Chem 287:16521\u0026ndash;16529\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCsermely P, Kunsic N, Mendik P, Kerestely M, Farago T, Veres DV, Tompa P (2020) Learning of Signaling Networks: Molecular Mechanisms. Trends Biochem Sci 45:284\u0026ndash;294\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDouglas JL, Trieber CA, Afara M, Young HS (2005) Rapid, high-yield expression and purification of Ca2+-ATPase regulatory proteins for high-resolution structural studies. Protein Expr Purif 40:118\u0026ndash;125\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEletr S, Inesi G (1972) Phospholipid orientation in sarcoplasmic membranes: spin-label ESR and proton MNR studies. Biochim Biophys Acta 282:174\u0026ndash;179\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFisher ME, Bovo E, Aguayo-Ortiz R, Cho EE, Pribadi MP, Dalton MP, Rathod N, Lemieux MJ, Espinoza-Fonseca LM, Robia SL, Zima AV, Young HS (2021) Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA. Elife 10:e65545\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGlaves JP, Primeau JO, Gorski PA, Espinoza-Fonseca LM, Lemieux MJ, Young HS (2020) Interaction of a Sarcolipin Pentamer and Monomer with the Sarcoplasmic Reticulum Calcium Pump, SERCA. Biophys J 118:518\u0026ndash;531\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGorski PA, Glaves JP, Vangheluwe P, Young HS (2013) Sarco(endo)plasmic reticulum calcium ATPase (SERCA) inhibition by sarcolipin is encoded in its luminal tail. J Biol Chem 288:8456\u0026ndash;8467\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGustavsson M, Verardi R, Mullen DG, Mote KR, Traaseth NJ, Gopinath T, Veglia G (2013) Allosteric regulation of SERCA by phosphorylation-mediated conformational shift of phospholamban. Proc Natl Acad Sci U S A 110:17338\u0026ndash;17343\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaeda S, Taketsugu T, Morokuma K (2012) Automated Exploration of Photolytic Channels of HCOOH: Conformational Memory via Excited-State Roaming. J Phys Chem Lett 3:1900\u0026ndash;1907\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMakarov DE (2021) Barrier Crossing Dynamics from Single-Molecule Measurements. J Phys Chem B 125:2467\u0026ndash;2476\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMall S, Broadbridge R, Harrison SL, Gore MG, Lee AG, East JM (2006) The presence of sarcolipin results in increased heat production by Ca(2+)-ATPase. J Biol Chem 281:36597\u0026ndash;36602\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNelson BR, Makarewich CA, Anderson DM, Winders BR, Troupes CD, Wu F, Reese AL, McAnally JR, Chen X, Kavalali ET, Cannon SC, Houser SR, Bassel-Duby R, Olson EN (2016) A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle. Science 351:271\u0026ndash;275\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOdermatt A, Becker S, Khanna VK, Kurzydlowski K, Leisner E, Pette D, MacLennan DH (1998) Sarcolipin regulates the activity of SERCA1, the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase. J Biol Chem 273:12360\u0026ndash;12369\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSahoo SK, Shaikh SA, Sopariwala DH, Bal NC, Bruhn DS, Kopec W, Khandelia H, Periasamy M (2015) The N Terminus of Sarcolipin Plays an Important Role in Uncoupling Sarco-endoplasmic Reticulum Ca2+-ATPase (SERCA) ATP Hydrolysis from Ca2\u0026thinsp;+\u0026thinsp;Transport. J Biol Chem 290:14057\u0026ndash;14067\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSahoo SK, Shaikh SA, Sopariwala DH, Bal NC, Periasamy M (2013) Sarcolipin protein interaction with sarco(endo)plasmic reticulum Ca2\u0026thinsp;+\u0026thinsp;ATPase (SERCA) is distinct from phospholamban protein, and only sarcolipin can promote uncoupling of the SERCA pump. J Biol Chem 288:6881\u0026ndash;6889\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchorner M, Beyer SR, Southall J, Cogdell RJ, Kohler J (2015) Conformational Memory of a Protein Revealed by Single-Molecule Spectroscopy. J Phys Chem B 119:13964\u0026ndash;13970\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeidel K, Andronesi OC, Krebs J, Griesinger C, Young HS, Becker S, Baldus M (2008) Structural characterization of Ca(2+)-ATPase-bound phospholamban in lipid bilayers by solid-state nuclear magnetic resonance (NMR) spectroscopy. Biochemistry 47:4369\u0026ndash;4376\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmeazzetto S, Armanious GP, Moncelli MR, Bak JJ, Lemieux MJ, Young HS, Tadini-Buoninsegni F (2017) Conformational memory in the association of the transmembrane protein phospholamban with the sarcoplasmic reticulum calcium pump SERCA. J Biol Chem 292:21330\u0026ndash;21339\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSorensen TL, Moller JV, Nissen P (2004) Phosphoryl transfer and calcium ion occlusion in the calcium pump. Science 304:1672\u0026ndash;1675\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStokes DL, Green NM (1990) Three-dimensional crystals of CaATPase from sarcoplasmic reticulum. Symmetry and molecular packing. Biophys J 57:1\u0026ndash;14\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTadini-Buoninsegni F (2020) Protein Adsorption on Solid Supported Membranes: Monitoring the Transport Activity of P-Type ATPases. Molecules 25\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTadini-Buoninsegni F, Bartolommei G, Moncelli MR, Guidelli R, Inesi G (2006) Pre-steady state electrogenic events of Ca2+/H+ exchange and transport by the Ca2+-ATPase. J Biol Chem 281:37720\u0026ndash;37727\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTadini-Buoninsegni F, Bartolommei G, Moncelli MR, Tal DM, Lewis D, Inesi G (2008) Effects of high-affinity inhibitors on partial reactions, charge movements, and conformational States of the Ca2\u0026thinsp;+\u0026thinsp;transport ATPase (sarco-endoplasmic reticulum Ca2\u0026thinsp;+\u0026thinsp;ATPase). Mol Pharmacol 73:1134\u0026ndash;1140\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTadini Buoninsegni F, Bartolommei G, Moncelli MR, Inesi G, Guidelli R (2004) Time-resolved charge translocation by sarcoplasmic reticulum Ca-ATPase measured on a solid supported membrane. Biophys J 86:3671\u0026ndash;3686\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian Y, Meng L, Zhang Z (2020) What is strain in neurodegenerative diseases? Cell Mol Life Sci 77:665\u0026ndash;676\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToyoshima C, Asahi M, Sugita Y, Khanna R, Tsuda T, MacLennan DH (2003) Modeling of the inhibitory interaction of phospholamban with the Ca2\u0026thinsp;+\u0026thinsp;ATPase. Proc Natl Acad Sci U S A 100:467\u0026ndash;472\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToyoshima C, Iwasawa S, Ogawa H, Hirata A, Tsueda J, Inesi G (2013) Crystal structures of the calcium pump and sarcolipin in the Mg2+-bound E1 state. Nature 495:260\u0026ndash;264\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToyoshima C, Mizutani T (2004) Crystal structure of the calcium pump with a bound ATP analogue. Nature 430:529\u0026ndash;535\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToyoshima C, Nakasako M, Nomura H, Ogawa H (2000) Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution. Nature 405:647\u0026ndash;655\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToyoshima C, Nomura H (2002) Structural changes in the calcium pump accompanying the dissociation of calcium. Nature 418:605\u0026ndash;611\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrieber CA, Afara M, Young HS (2009) Effects of phospholamban transmembrane mutants on the calcium affinity, maximal activity, and cooperativity of the sarcoplasmic reticulum calcium pump. Biochemistry 48:9287\u0026ndash;9296\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrieber CA, Douglas JL, Afara M, Young HS (2005) The effects of mutation on the regulatory properties of phospholamban in co-reconstituted membranes. Biochemistry 44:3289\u0026ndash;3297\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTripathi T, Uversky VN, Giuliani A (2025) Intelligent' proteins. Cell Mol Life Sci 82:239\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWinther AM, Bublitz M, Karlsen JL, Moller JV, Hansen JB, Nissen P, Buch-Pedersen MJ (2013) The sarcolipin-bound calcium pump stabilizes calcium sites exposed to the cytoplasm. Nature 495:265\u0026ndash;269\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoung HS, Jones LR, Stokes DL (2001) Locating phospholamban in co-crystals with Ca(2+)-ATPase by cryoelectron microscopy. Biophys J 81:884\u0026ndash;894\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoung HS, Rigaud JL, Lacapere JJ, Reddy LG, Stokes DL (1997) How to make tubular crystals by reconstitution of detergent-solubilized Ca2(+)-ATPase. Biophys J 72:2545\u0026ndash;2558\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"the-journal-of-membrane-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmbi","sideBox":"Learn more about [The Journal of Membrane Biology](http://link.springer.com/journal/232)","snPcode":"232","submissionUrl":"https://submission.nature.com/new-submission/232/3","title":"The Journal of Membrane Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Sarcoplasmic reticulum, calcium transport, charge translocation, ATPase activity, P-type ATPase, regulatory peptide","lastPublishedDoi":"10.21203/rs.3.rs-9544726/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9544726/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMuscle relaxation is enabled by the sarco-endoplasmic reticulum Ca\u003csup\u003e2+\u003c/sup\u003e- ATPase (SERCA), which removes calcium from the cytosol and returns it to the lumen of the sarcoplasmic reticulum. During transport, SERCA adopts a variety of conformational states which differ in their structure and affinity for substrates. The transition between these states is mediated by calcium and ATP binding and the formation of an aspartyl-phosphate intermediate, which enables the transport of calcium across the membrane. SERCA function is highly regulated because of the importance of calcium in processes such as muscle contraction-relaxation. A family of tissue-specific transmembrane regulatory subunits interact with SERCA, exemplified by sarcolipin (SLN) in skeletal muscle and phospholamban (PLN) in cardiac muscle. SLN and PLN are known to alter the apparent calcium affinity and maximal activity of SERC. In the present study, we investigated SLN inhibition of SERCA under conditions that varied the substrate-dependent conformational state of SERCA. Measuring both calcium-dependent ATP hydrolysis and charge translocation, we found that SLN inhibition was dependent on the initial state of SERCA. Under substrate conditions that poised SERCA in the calcium-free E2 state, SLN was more inhibitory and impacted both the maximal activity and apparent calcium affinity of SERCA. In contrast, SLN inhibition was reduced under pre-incubation conditions that favored the calcium-bound E1 state of SERCA. We conclude that SLN is capable of distinct modes of interaction with SERCA depending on the conformational state, and that the mode of interaction exhibits conformational memory in that the initial state persists during steady-state turnover of SERCA.\u003c/p\u003e","manuscriptTitle":"Sarcolipin is a conformation-dependent regulator of the sarcoplasmic reticulum calcium pump SERCA","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-07 10:57:25","doi":"10.21203/rs.3.rs-9544726/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-08T15:05:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"231118368702555300931195823502383084313","date":"2026-04-30T08:55:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-29T12:22:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-28T10:22:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-28T10:22:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"The Journal of Membrane Biology","date":"2026-04-27T17:01:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"the-journal-of-membrane-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmbi","sideBox":"Learn more about [The Journal of Membrane Biology](http://link.springer.com/journal/232)","snPcode":"232","submissionUrl":"https://submission.nature.com/new-submission/232/3","title":"The Journal of Membrane Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0f4a9515-1f52-452f-8248-520620a55175","owner":[],"postedDate":"May 7th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-08T15:05:37+00:00","index":15,"fulltext":""},{"type":"reviewerAgreed","content":"231118368702555300931195823502383084313","date":"2026-04-30T08:55:30+00:00","index":11,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-07T10:57:26+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-07 10:57:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9544726","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9544726","identity":"rs-9544726","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}