The Ca2+ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca2+ in intracellular membrane bound compartments, thereby lowering cytosolic Ca2+ to induce relaxation. The stored Ca2+ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca2+, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca2+. We review here biochemical and biophysical evidence demonstrating that release of bound Ca2+ into the lumen of SR requires Ca2+/H+ exchange at the low affinity Ca2+ sites. Rise of lumenal Ca2+ above its dissociation constant from low affinity sites, or reduction of the H+ concentration by high pH, prevent Ca2+/H+ exchange. Under these conditions Ca2+ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca2+pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.
Sarcoplasmic reticulum (SR) membrane vesicles, originally referred to as “relaxing factor”, were first isolated from skeletal muscle by Ebashi and Lipmann (1962), and Hasselbach and Makinose (1962), and were shown to contain a P–type ATPase (SERCA1 isoform) sustaining Ca2+ transport. In muscle cells, this transport activity plays an important role in lowering cytosolic Ca2+ as required for relaxation of contractile elements, and storing transported Ca2+ in the lumen of SR for subsequent release and contractile activation (Carafoli 2002; Clapham 2007). General information on SERCA1 catalytic function and molecular structure is given in several reviews (de Meis and Vianna 1979; Inesi et al. 1990; Andersen and Vilsen 1995; Toyoshima 2008; Møller et al. 2010).
SERCA1 is a 996 amino acid membrane bound protein (MacLennan et al. 1985) comprising ten transmembrane helical segments, and a globular headpiece that protrudes from the cytosolic side of the membrane and includes three distinct domains (A, N and P). Catalytic activation follows high affinity binding of cytosolic Ca2+ within the transmembrane region of the enzyme (Fig. 1). Activation is followed by utilization of ATP bound to the N domain, and formation of phosphorylated enzyme intermediate by transfer of the ATP γ-phosphate to an aspartyl residue (Asp-351) in the P domain. Conformational transition of the phosphoenzyme then promotes vectorial translocation of bound Ca2+ and release of Ca2+ into the lumen of SR. Finally, the phosphoenzyme undergoes hydrolytic cleavage with catalytic assistance by an A domain critical sequence (Thr-Gly-Glu), leading to a new cycle.
Ca2+/ATP coupling ratios
Cooperative binding of 2 Ca2+ per ATPase (Inesi et al. 1980) implies transport of 2 Ca2+ per catalytic cycle, if both bound Ca2+ are translocated with maximal efficiency. Ratios of 2 Ca2+ per ATP were in fact observed under conditions permitting free Ca2+ to remain low in the lumen of the vesicles: (a) steady state experiments in which oxalate is used for complexation of lumenal Ca2+ (Martonosi and Feretos 1964) and (b) pre-steady state experiments in which lumenal Ca2+ has yet to rise (Fig. 2a; Inesi et al. 1988). On the other hand, Ca2+/ATP ratios lower than 2 have been observed with native SR vesicles as well as reconstituted systems (Yu and Inesi 1995), under conditions permitting lumenal Ca2+ to rise (mM) while Ca2+ in the outer medium remains sufficiently high (μM) for ATPase activation (Fig. 2b). Under these conditions, the lumenal Ca2+ concentration is higher than the dissociation constant of Ca2+ from the lumenal sites, and therefore the phosphoenyme bypasses the Ca2+ release step and proceeds to hydrolytic cleavage of Pi, with consequent reduction of the Ca2+/ATP transport ratio. Uncoupled ATPase subsides if EGTA is added to the outer medium to reduce free Ca2+ below the ATPase activating level (Fig. 2b).
A variable stoichiometric ratio (i.e., Ca2+/ATP) of active transport may be considered to be an intrinsic feature of the pump, if the ATPase reaction sequence allows an alternate pathway leading to hydrolytic cleavage of Pi without vectorial displacement of Ca2+ (Johnson et al. 1985; Inesi and de Meis 1989). The importance of this phenomenon, referred to as slippage of the pump, is related to heat production and thermogenesis, when the free energy derived from ATP hydrolysis is not utilized for active transport (de Meis et al. 1997; de Meis 2001; de Meis et al. 2005).
Ca2+/H+ exchange at the lumenal gate
Exchange of Ca2+ with H+ upon vectorial translocation is a specific feature of the Ca2+ ATPase (Lewis et al. 2012), facilitating lumenal Ca2+ release (Yu et al. 1994; Bublitz et al. 2013). Evidence of Ca2+/H+ exchange, H+ counter transport (Chiesi and Inesi 1980; Yamaguchi and Kanazawa 1985; Ueno and Sekine 1981) and electrogenicity (Morimoto and Kasai 1986; Cornelius and Møller 1991; Obara et al. 2005) in the operation of the Ca2+ ATPase was obtained with vesicular fragments of SR membrane and with ATPase reconstituted in phospholipids vesicles lacking non specific H+ or Ca2+ channels. It is shown in Fig. 3a that the molar ratio of Ca2+/H+ counter transport is 1 when the lumenal and medium pH is near neutrality. However, a higher number of acidic residues involved in Ca2+ binding (Glu-771, Asp-800, Glu-309, Glu-908) is likely to participate in Ca2+/H+ exchange (Bublitz et al. 2013; Obara et al. 2005), even though only one H+ per Ca2+ may actually be counter transported. In this case, the remaining H+ undergo lumenal dissociation. The Ca2+/H+ exchange is facilitated by acidic residues pK changes, as the phosphoenzyme undergoes its catalytic transition (Yu et al. 1994).
Further evidence for Ca2+/H+ exchange is provided by measurements of charge transfer upon addition of Ca2+ or ATP to microsomal vesicles adsorbed on a solid supported membrane (SSM) (Tadini-Buoninsegni et al. 2004, 2006, 2010). Related electrogenic events are recorded as current transients due to flow of electrons along the external circuit toward the electrode surface, as required to compensate for the potential difference across the vesicular membrane produced by displacement of positive charge upon vectorial translocation in the direction of the SSM electrode. In fact, when ATP is added to the membrane bound ATPase adsorbed on the SSM in the presence of saturating Ca2+, a current transient is observed due to vectorial translocation and dissociation of bound Ca2+ in the direction of the SSM electrode after phosphoenzyme formation by utilization of ATP (Tadini-Buoninsegni et al. 2004, 2006). The electrical current recorded by the SSM method is a measure of the rate of change of the transmembrane potential and is not sensitive to stationary currents. Therefore, only the electrogenic signal generated within the first cycle is observed, whereas steady state events after the first cycle are not detected. It is shown in Fig. 3b that the net charge produced by ATP addition at neutral pH decreases significantly if ATP addition is performed at alkaline pH. This indicates that when lack of H+ limits H+/Ca2+ exchange (i.e. alkaline pH), vectorial translocation of bound Ca2+ in the direction of the SSM electrode is prevented, even though K+ is present in high concentration and may neutralize acidic residues at alkaline pH. This indicates a requirement for specific H+ binding at the Ca2+ sites, in order to obtain Ca2+ release.
The specific relevance of ATP dependent charge transfer is demonstrated by interference of mutations (Asp-351 to Asn) preventing phosphoenzyme formation (Tadini-Buoninsegni et al. 2006). Furthermore, cation/H+ exchange at the transport sites following phosphoenzyme formation occurs in Ca2+ ATPases, but does not occur in copper ATPases (Lewis et al. 2012).
The importance of Ca2+/H+ exchange in determining release of bound Ca2+ from the phosphoenzyme can be also demonstrated in steady state experiments. It is shown in Fig. 4 that the maximal levels of accumulated Ca2+ are significantly reduced if the pH is raised above 7 (consider that the physiological intracellular pH is 6.8, while the extracellular pH is 7.4)). This indicates that if exchange is limited due to low H+ concentration, Ca2+ is less likely to dissociate from the phosphoenzyme. On the other hand, while Ca2+ translocation is reduced, steady state ATPase activity is increased as the pH is raised, and continues after maximal levels of Ca2+ uptake are reached. It is apparent that alkaline pH reduces Ca2+/H+ exchange and dissociation of bound Ca2+, whereby the phosphoenyme bypasses the Ca2+ release step and proceeds to hydrolytic cleavage of Pi. Therefore, reduction of the Ca2+/ATP transport ratio can be produced either by a high Ca2+ concentration or a low H+ concentration in the lumen of the vesicles.
A diagram of the sequential steps in the ATPase mechanism, derived from the original reaction diagram of de Meis and Vianna (1979) and modified to show Ca2+/H+ exchange and a pathway for slippage of the Ca2+ pump, is given in Scheme 1.
In the diagram on Scheme 1, solid lines indicate the optimal pathway, beginning with enzyme activation by high affinity and cooperative binding of two Ca2+, yielding E1 · 2Ca2+. Utilization of ATP yields ADP · E1 ~ P · 2Ca2+, followed by release of ADP and utilization of the phosphorylation potential to change vectorial orientation and affinity of the Ca2+ sites. Bound Ca2+ is then released into the lumenal medium in exchange for H+. Hydrolytic cleavage of nH+ · E2-P and transition of nH+ · E2 to E1 finally yields closure of the lumenal gate and exposure of the Ca2+ sites to the cytosolic medium. Formation of E1 · 2Ca2+ then starts a new cycle.
The dotted lines in Scheme 1 indicate that if lumenal Ca2+ is higher than its dissociation constant, or lumenal H+ is too low to sustain exchange, Ca2+ release and formation of nH+ · E2 are prevented. Interference with completion of the ATPase cycle would then cause reversal to E1 ~ P · 2Ca2+ (see below Fig. 5b), whereby phosphorylation potential leads directly to hydrolytic cleavage, rather than utilization for active transport. This is rendered possible as low concentration of ADP prevents its re-binding, and the remaining proximity of the A domain to the phosphorylation site allows catalytic assistance by the critical Thr-Gly-Glu sequence.
Strong evidence for the role of protons and the nH+ · E2 state is provided with experiments of enzyme phosphorylation by utilization of Pi (Masuda and de Meis 1973). It is shown in Fig. 5a that this reaction (i.e., reverse reaction of phosphoenzyme hydrolytic cleavage) is enhanced by acid pH, and is inhibited by alkaline pH and Ca2+. This indicates a requirement for proton occupancy of acidic residues involved in Ca2+ binding (i.e., nH+ · E2). On the other hand (Fig. 5b), further reversal of the cycle upon addition of ADP to form ATP, requires a switch to alkaline pH and addition of mM Ca2+, in order to replace protons with Ca2+ on the low affinity binding sites (i.e. transition of nH+ · E2-P to E2-P · 2Ca2+ and ADP · E1 ~ P · 2Ca2+).
High resolution crystal structures of most states (or their analogs) comprising the ATPase reaction sequence have been obtained, and are described in detailed reviews (Toyoshima 2008; Møller et al. 2010; Toyoshima and Inesi 2004). Comparison of these structures reveals rearrangements of transmembrane helices upon Ca2+ binding, phosphoenzyme formation, occlusion and then dissociation of bound Ca2+, which are mechanically linked to specific bending and rotation patterns of each headpiece domain. These movements provide an explanation for the long range linkage of phosphorylation and Ca2+ binding domains, including the roles of critical amino acids in substrate binding, catalytic reactions, and Ca2+ transport. They also demonstrate that the states included in the reaction diagram are structurally distinct, and possess specific features that are functionally relevant. It is of interest that movement of M1-M2 causes displacement of membrane helix M4L from M5 and M6, thereby opening the lumenal gate and allowing outflow of Ca2+, following Ca2+/H+ exchange. The lumenal gate is then closed upon cleavage of phosphate and dissociation of H+, when reverse rotation of the A domain is accompanied by upward displacement of M4L and reduction of the space between the M4 and M6 helices.
Effects of accessory polypeptides
Several studies have demonstrated that sarcolipin (SLN), a 31 amino acid polypeptide (Odermatt et al. 1997; Odermatt et al. 1998) is constitutively bound to the Ca2+ ATPase (SERCA1) of (at least human and rabbit) fast twitch skeletal muscle, and produces uncoupling of ATP utilization and Ca2+ transport, with a consequent thermogenic effect (Mall et al. 2006; Bal et al. 2012). However, these studies were performed by genetic manipulations and reconstitution procedures, which may not apply to the physiologic signaling mechanism of a fast twitch in native muscles, but rather reflect phenomena occurring under special circumstance and/or in other tissues (see below). In fact, other studies have shown that incorporation of SLN into proteoliposomes with SERCA simply results in a lower apparent affinity for calcium and a lower turnover rate (Gorski et al. 2013).
It is of interest that the Ca2+ ATPase SERCA2 isoform, prevalent in cardiac muscle (Lytton et al. 1992), is associated with phospholamban (PLN), a 52 amino acid polypeptide, to some extent similar to SLN. The definite effect of PLB on SERCA2 is a lower Ca2+ binding affinity (Koss and Kranias 1996; MacLennan and Kranias 2003; Toyoshima et al. 2003) and/or a slower E1 to 2Ca2+ · E1 transition (Cantilina et al. 1993). This results in a higher Ca2+ concentration requirement for Ca2+ transport activation (Fig. 6).
Both SLN and PLN reside within a groove surrounded by transmembrane helices M2, M4, M6 and M9, as shown by crystallographic studies (Toyoshima et al. 2013; Winther et al. 2013; Akin et al. 2013), and also indicated by NMR (Buffy et al. 2006) and cross-linking experiments (Sahoo et al. 2013). This is a critical position, since structural studies demonstrate that the helices delimiting this groove undergo displacements affecting Ca2+ binding, Ca2+ dissociation, as well as opening and closing of the lumenal gate. This explains how the presence of SLN and PLN may affect rates of movements and related partial reactions of the ATPase cycle. Some difference in the effects of the two polypeptides could be related to specific sequences and points of interactions (Sahoo et al. 2013), as well as to the cytosolic segment of PLN (absent in SLN) which may interact with SERCA headpiece domains and delay their movements to yield the E1 .2Ca2+ state. It is worth considering that a slight shift of the Ca2+ concentration required for ATP activation would affect the rates of Ca2+ transport at low levels of cytoplasmic Ca2+, and therefore the efficiency of twitch relaxation. However, it is not likely that the Km (equilibrium constant) of the Ca2+ sites on lumenal orientation would be significantly affected. The physiological interest of PLB and SLN is related to the reversibility of their effects upon phosphorylation catalyzed by signaling kinases (Koss and Kranias 1996; MacLennan and Kranias 2003; Toyoshima et al. 2003).
Contractile relaxation and uncoupling of the Ca2+ pump
Considering the possibility of Ca2+ pump uncoupling, an important question is whether uncoupling of SERCA1 interferes with reduction of cytosolic Ca2+ below the level allowing contractile relaxation of muscle fibers. The evidence presented above indicates that the Ca2+ pump is perfectly coupled when the SR lumen Ca2+ in is low, even if cytosolic Ca2+ out is relatively high. In the light of this information, we consider that relaxation of a muscle twitch occurs in less than 1 s and, within this time, lumenal Ca2+ does not reach a concentration higher than its dissociation from E2-P · 2Ca2+, as shown in experiments performed with rabbit native SR vesicles (Fig. 2). On the other hand, when cytosolic Ca2+ is reduced to a level producing contractile relaxation, such a Ca2+ level will be also low with regard to SERCA activation, and the ATPase would then proceed at very low rates or remain inactive. Therefore during relaxation, the pump remains quiescent, mostly in the Mg2+ bound E1 state (Toyoshima et al. 2013), with no significant slippage. Slippage of the pump would occur if lumenal Ca2+ were to become higher than its dissociation constant from E2-P · 2Ca2+, and cytosolic Ca2+ were to remain above the level required for full ATPase activation. This may occur upon prolonged muscle activity, if cytosolic Ca2+ is maintained relatively high by multiple action potentials and Ca2+ flux through plasma membrane voltage sensitive channels, as expected in shivering thermogenesis. Alternatively, a rise of intracellular pH above 7.0, may affect intracellular Ca2+ signaling, as recently reported for G protein signaling (Isom et al. 2013). In this case, some degree of SERCA uncoupling would be produced, contributing to the muscle twitching observed in alkalosis. Furthermore, thermogenic uncoupling may occur in tissues where SERCA is inserted in membrane compartments allowing lumenal Ca2+ rise to high levels, while cytosolic Ca2+ remains sufficiently high. Most importantly, it was reported that in brown fat, in addition to uncoupling of the mitochondrial respiratory chain, uncoupled SERCA contributes to non shivering thermogenesis (de Meis et al. 2006).
Sarcoplasmic reticulum Ca2+ ATPase
Solid supported membrane
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The authors were partially supported by a National Institutes of Health RO1 Grant (G.I.), and Ente Cassa di Risparmio di Firenze and Italian Ministry of Education, University and Research (F.T.-B.).
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Inesi, G., Tadini-Buoninsegni, F. Ca2+/H+ exchange, lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase. J. Cell Commun. Signal. 8, 5–11 (2014) doi:10.1007/s12079-013-0213-7
- SERCA Ca2+ATPase
- Ca2+/ATP coupling ratios
- Ca2+/H+ exchange
- Ca2+ signaling