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The Ca 2+ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca 2+ in intracellular membrane bound compartments, thereby lowering cytosolic Ca 2+ to induce relaxation. The stored Ca 2+ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca 2+ , whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca 2+ . We review here biochemical and biophysical evidence demonstrating that release of bound Ca 2+ into the lumen of SR requires Ca 2+ /H + exchange at the low affinity Ca 2+ sites. Rise of lumenal Ca 2+ above its dissociation constant from low affinity sites, or reduction of the H + concentration by high pH, prevent Ca 2+ /H + exchange. Under these conditions Ca 2+ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca 2+ pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.

$Ca^{2+}-ATPase$of sarcoplasmic reticulum is an ATP-powered Ca2+pump but also a H+pump in the opposite direction with no demonstrated functional role. Here, we report a$2.4-\\ring{A}-resolution$crystal structure of the$Ca^{2+}-ATPase$in the absence of Ca2+stabilized by two inhibitors, dibutyldihydroxybenzene, which bridges two transmembrane helices, and thapsigargin, also bound in the membrane region. Now visualized are water and several phospholipid molecules, one of which occupies a cleft between two transmembrane helices. Atomic models of the Ca2+binding sites with explicit hydrogens derived by continuum electrostatic calculations show how water and protons fill the space and compensate charge imbalance created by$Ca^{2+}-release$. They suggest that H+countertransport is a consequence of a requirement for maintaining structural integrity of the empty Ca2+-binding sites. For this reason, cation countertransport is probably mandatory for all P-type ATPases and possibly accompanies transport of water as well.

The X-ray crystal structures of SERCA1a, a Ca 2+ -ATPase from the sarcoplasmic reticulum, in the presence and absence of sarcolipin are reported; the structures indicate that sarcolipin stabilizes SERCA1a in an ‘open’ state that has not been well characterised previously, in which SERCA1a has not yet accepted calcium into its two high-affinity binding sites. How calcium drives muscle cells Muscle cell contraction and relaxation are controlled by the rise and fall of cytosolic calcium concentrations, initiated by the release of Ca 2+ from the sarcoplasmic reticulum (SR) and terminated by its re-sequestration by the SR Ca 2+ -ATPase (SERCA). Two papers in this issue of Nature present the X-ray crystal structures of SERCA in the presence of sarcolipin, a small membrane protein that regulates SERCA in skeletal muscle. The structures indicate that sarcolipin traps SERCA in a previously unknown 'open' state, in which SERCA has not yet accepted calcium into its two high-affinity binding sites. P-type ATPases are ATP-powered ion pumps that establish ion concentration gradients across biological membranes, and are distinct from other ATPases in that the reaction cycle includes an autophosphorylation step. The best studied is Ca 2+ -ATPase from muscle sarcoplasmic reticulum (SERCA1a), a Ca 2+ pump that relaxes muscle cells after contraction, and crystal structures have been determined for most of the reaction intermediates 1 , 2 . An important outstanding structure is that of the E1 intermediate, which has empty high-affinity Ca 2+ -binding sites ready to accept new cytosolic Ca 2+ . In the absence of Ca 2+ and at pH 7 or higher, the ATPase is predominantly in E1, not in E2 (low affinity for Ca 2+ ) 3 , and if millimolar Mg 2+ is present, one Mg 2+ is expected to occupy one of the Ca 2+ -binding sites with a millimolar dissociation constant 4 , 5 . This Mg 2+ accelerates the reaction cycle 4 , not permitting phosphorylation without Ca 2+ binding. Here we describe the crystal structure of native SERCA1a (from rabbit) in this E1·Mg 2+ state at 3.0 Å resolution in addition to crystal structures of SERCA1a in E2 free from exogenous inhibitors, and address the structural basis of the activation signal for phosphoryl transfer. Unexpectedly, sarcolipin 6 , a small regulatory membrane protein of Ca 2+ -ATPase 7 , is bound, stabilizing the E1·Mg 2+ state. Sarcolipin is a close homologue of phospholamban, which is a critical mediator of β-adrenergic signal in Ca 2+ regulation in heart (for reviews, see, for example, refs 8–10 ), and seems to play an important role in muscle-based thermogenesis 11 . We also determined the crystal structure of recombinant SERCA1a devoid of sarcolipin, and describe the structural basis of inhibition by sarcolipin/phospholamban. Thus, the crystal structures reported here fill a gap in the structural elucidation of the reaction cycle and provide a solid basis for understanding the physiological regulation of the calcium pump.

The structures of the Ca2+-ATPase (SERCA1a) have been determined for five different states by X-ray crystallography. Detailed comparison of the structures in the Ca2+ bound form and unbound (but thapsigargin bound) form reveals that very large rearrangements of the transmembrane helices take place accompanying Ca2+ dissociation and binding and that they are mechanically linked with equally large movements of the cytoplasmic domains. The meanings of the rearrangements of the transmembrane helices and those of the cytoplasmic domains as well as the mechanistic roles of phosphorylation are now becoming clear. Furthermore, the roles of critical amino acid residues identified by extensive mutagenesis studies are becoming evident in terms of atomic structure. [PUBLICATION ABSTRACT]

The Ca(2+) transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca(2+) in intracellular membrane bound compartments, thereby lowering cytosolic Ca(2+) to induce relaxation. The stored Ca(2+) is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca(2+), whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca(2+). We review here biochemical and biophysical evidence demonstrating that release of bound Ca(2+) into the lumen of SR requires Ca(2+)/H(+) exchange at the low affinity Ca(2+) sites. Rise of lumenal Ca(2+) above its dissociation constant from low affinity sites, or reduction of the H(+) concentration by high pH, prevent Ca(2+)/H(+) exchange. Under these conditions Ca(2+) release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca(2+)pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.

The Ca(2+) transport ATPase of intracellular membranes (SERCA) can be inhibited by a series of chemical compounds such as Thapsigargin (TG), 2,5-di(tert-butyl)hydroquinone (DBHQ) and 1,3-dibromo-2,4,6-tris (methyl-isothio-uronium) benzene (Br(2)-TITU). These compounds have specific binding sites in the ATPase protein, and different mechanisms of inhibition. On the other hand, SERCA gene silencing offers a convenient and specific method for suppression of SERCA activity in cells. The physiological and pharmacological implications of SERCA inhibition are discussed.

The calcium transport ATPase and the copper transport ATPase are members of the P-ATPase family and retain an analogous catalytic mechanism for ATP utilization, including intermediate phosphoryl transfer to a conserved aspartyl residue, vectorial displacement of bound cation, and final hydrolytic cleavage of Pi. Both ATPases undergo protein conformational changes concomitant with catalytic events. Yet, the two ATPases are prototypes of different features with regard to transduction and signaling mechanisms. The calcium ATPase resides stably on membranes delimiting cellular compartments, acquires free Ca 2+ with high affinity on one side of the membrane, and releases the bound Ca 2+ on the other side of the membrane to yield a high free Ca 2+ gradient. These features are a basic requirement for cellular Ca 2+ signaling mechanisms. On the other hand, the copper ATPase acquires copper through exchange with donor proteins, and undergoes intracellular trafficking to deliver copper to acceptor proteins. In addition to the cation transport site and the conserved aspartate undergoing catalytic phosphorylation, the copper ATPase has copper binding regulatory sites on a unique N-terminal protein extension, and has also serine residues undergoing kinase assisted phosphorylation. These additional features are involved in the mechanism of copper ATPase intracellular trafficking which is required to deliver copper to plasma membranes for extrusion, and to the trans-Golgi network for incorporation into metalloproteins. Isoform specific glyocosylation contributes to stabilization of ATP7A copper ATPase in plasma membranes.

Sarcoplasmic reticulum vesicles were adsorbed on an octadecanethiol/phosphatidylcholine mixed bilayer anchored to a gold electrode, and the Ca-ATPase contained in the vesicles was activated by ATP concentration jumps both in the absence and in the presence of K + ions and at different pH values. Ca 2+ concentration jumps in the absence of ATP were also carried out. The resulting capacitive current transients were analyzed together with the charge under the transients. The relaxation time constants of the current transients were interpreted on the basis of an equivalent circuit. The current transient after ATP concentration jumps and the charge after Ca 2+ concentration jumps in the absence of ATP exhibit almost the same dependence upon the Ca 2+ concentration, with a half-saturating value of ∼1.5 μM. The pH dependence of the charge after Ca 2+ translocation demonstrates the occurrence of one H + per one Ca 2+ countertransport at pH 7 by direct charge-transfer measurements. The presence of K + decreases the magnitude of the current transients without altering their shape; this decrease is explained by K + binding to the cytoplasmic side of the pump in the E 1 conformation and being released to the same side during the E 1–E 2 transition.

Involvement of the calcineurin/NFAT pathway in transcription of cardiac sarcoplasmic reticulum Ca 2+ ATPase (SERCA2) was demonstrated (Prasad and Inesi, Am J Physiol Heart Circ Physiol 300(1):H173–H180, 2011) by upregulation of SERCA2 following calcineurin (CN) activation by cytosolic Ca 2+ , and downregulation of SERCA2 following CN inhibition with cyclosporine (CsA) or CN subunits gene silencing. We show here that in cultured cardiac myocytes, competitive engagement of the CN/NFAT pathway is accompanied by downregulation of SERCA2 and Ca 2+ signaling alterations. In fact, SERCA2 downregulation occurs following infection of myocytes with adenovirus vectors carrying luciferase or SERCA1 cDNA under control of NFAT-dependent promoters, but not under control of CMV promoters that do not depend on NFAT. SERCA2 downregulation is demonstrated by comparison with endogenous transcription and protein expression standards such as GAPDH and actin, indicating prominent SERCA2 involvement by the CN/NFAT pathway. Transcription of genes involved in hypertrophy, triggered by adrenergic agonist or by direct protein kinase C (PKC) activation with phorbol 12-myristate 13-acetate (PMA), is also prominently dependent on CN/NFAT. This is demonstrated by CN inhibition with CsA, CN subunits gene silencing with siRNA, displacement of NFAT from CN with 9,10-Dihydro-9,10[1′,2′]-benzenoanthracene-1,4-dione (INCA-6), and myocyte infection with vectors carrying luciferase cDNA under control of NFAT-dependent promoter. We show here that competitive engagement of the CN/NFAT pathway by endogenous genes involved in hypertrophy produces downregulation of SERCA2, reduction of Ca 2+ transport and inadequate Ca 2+ signaling. It is most interesting that, in the presence of adrenergic agonist, specific protein kinase C (PKC) inhibition with 3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione (Gö 6983) prevents development of hypertrophy and maintains adequate SERCA2 levels and Ca 2+ signaling.

Ca(2+)-ATPase of sarcoplasmic reticulum is an ATP-powered Ca(2+) pump but also a H(+) pump in the opposite direction with no demonstrated functional role. Here, we report a 2.4-A-resolution crystal structure of the Ca(2+)-ATPase in the absence of Ca(2+) stabilized by two inhibitors, dibutyldihydroxybenzene, which bridges two transmembrane helices, and thapsigargin, also bound in the membrane region. Now visualized are water and several phospholipid molecules, one of which occupies a cleft between two transmembrane helices. Atomic models of the Ca(2+) binding sites with explicit hydrogens derived by continuum electrostatic calculations show how water and protons fill the space and compensate charge imbalance created by Ca(2+)-release. They suggest that H(+) countertransport is a consequence of a requirement for maintaining structural integrity of the empty Ca(2+)-binding sites. For this reason, cation countertransport is probably mandatory for all P-type ATPases and possibly accompanies transport of water as well.