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 ca transport . in muscle cells , this transport activity plays an important role in lowering cytosolic ca as required for relaxation of contractile elements , and storing transported ca 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 ; mller et al . 2010 ) . 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 ca 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 -phosphate to an aspartyl residue ( asp-351 ) in the p domain . conformational transition of the phosphoenzyme then promotes vectorial translocation of bound ca and release of ca 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.fig . the diagram shows ten transmembrane segments ( m1 to m10 ) including six residues ( glu-309 , glu-771 , asn-796 , thr-799 , asp-800 and glu-908 ) contributing oxygen atoms for calcium binding , enzyme activation , and transport . the extramembranous headpiece comprises : a nucleotide binding domain ( n ) ; the p domain , with several residues conserved in p - type atpases , including asp-351 ( in red ) that undergoes phosphorylation to form the catalytic phosphoenzyme intermediate ( ep ) ; and the a domain with the thr - gly - glu conserved sequence involved in catalytic assistance of ep hydrolytic cleavage two - dimensional folding model of the serca1 sequence . the diagram shows ten transmembrane segments ( m1 to m10 ) including six residues ( glu-309 , glu-771 , asn-796 , thr-799 , asp-800 and glu-908 ) contributing oxygen atoms for calcium binding , enzyme activation , and transport . the extramembranous headpiece comprises : a nucleotide binding domain ( n ) ; the p domain , with several residues conserved in p - type atpases , including asp-351 ( in red ) that undergoes phosphorylation to form the catalytic phosphoenzyme intermediate ( ep ) ; and the a domain with the thr - gly - glu conserved sequence involved in catalytic assistance of ep hydrolytic cleavage 1980 ) implies transport of 2 ca per catalytic cycle , if both bound ca are translocated with maximal efficiency . ratios of 2 ca per atp were in fact observed under conditions permitting free ca to remain low in the lumen of the vesicles : ( a ) steady state experiments in which oxalate is used for complexation of lumenal ca ( martonosi and feretos 1964 ) and ( b ) pre - steady state experiments in which lumenal ca has yet to rise ( fig .  2a ; inesi et al . 1988 ) . on the other hand , ca / 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 ca to rise ( mm ) while ca in the outer medium remains sufficiently high ( m ) for atpase activation ( fig . the lumenal ca concentration is higher than the dissociation constant of ca from the lumenal sites , and therefore the phosphoenyme bypasses the ca release step and proceeds to hydrolytic cleavage of pi , with consequent reduction of the ca / atp transport ratio . uncoupled atpase subsides if egta is added to the outer medium to reduce free ca below the atpase activating level ( fig . 2pre - steady state measurements of atpase activity and ca transport by native sr vesicles obtained from skeletal muscle . a initial phosphoenzyme formation and ca occlusion ( 2ca/1ep ) b pre - steady state experiments extended to the second time scale , show that the initial rates of ca uptake and pi production begin with a ratio of 2:1 , but the ca uptake rate undergoes saturation , while uncoupled atpase activity continues as long as the medium ca is maintained above the atpase activation level . reaction mixtures contained 2050  g sr protein / ml , 10  mm pipes , ph  7.0 , 100  mm kcl , 5  mm mgso4 , 0.2  mm cacl2 and 0.2  mm egta . reaction started with 100  mm atp and stopped by acid quenching . 1  mm egta added when indicated . ( 1988 ) and yu and inesi ( 1995 ) pre - steady state measurements of atpase activity and ca transport by native sr vesicles obtained from skeletal muscle . a initial phosphoenzyme formation and ca occlusion ( 2ca/1ep ) b pre - steady state experiments extended to the second time scale , show that the initial rates of ca uptake and pi production begin with a ratio of 2:1 , but the ca uptake rate undergoes saturation , while uncoupled atpase activity continues as long as the medium ca is maintained above the atpase activation level . reaction mixtures contained 2050  g sr protein / ml , 10  mm pipes , ph  7.0 , 100  mm kcl , 5  mm mgso4 , 0.2  mm cacl2 and 0.2  mm egta . reaction started with 100  mm atp and stopped by acid quenching . 1  mm egta added when indicated . ( 1988 ) and yu and inesi ( 1995 ) a variable stoichiometric ratio ( i.e. , ca / 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 ca ( johnson et al . 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 . exchange of ca with h upon vectorial translocation is a specific feature of the ca atpase ( lewis et al . 2012 ) , facilitating lumenal ca release ( yu et al . 1994 ; bublitz et al . evidence of ca / 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 mller 1991 ; obara et al . 2005 ) in the operation of the ca atpase was obtained with vesicular fragments of sr membrane and with atpase reconstituted in phospholipids vesicles lacking non specific h or ca channels . 3a that the molar ratio of ca / h counter transport is 1 when the lumenal and medium ph is near neutrality . however , a higher number of acidic residues involved in ca binding ( glu-771 , asp-800 , glu-309 , glu-908 ) is likely to participate in ca / h+ exchange ( bublitz et al . 2005 ) , even though only one h per ca may actually be counter transported . in this case the ca / h exchange is facilitated by acidic residues pk changes , as the phosphoenzyme undergoes its catalytic transition ( yu et al . 1994).fig . 3 
 a atp - dependent ca uptake , h countertransport , and development of transmembrane electrical potential in reconstituted serca1 proteoliposomes . proteoliposomes prepared at ph  7.0 were diluted ( 5.0  g protein / ml ) in a medium ( ph  7.0 ) containing 10  mm pipes , 100  mm k2so4 , 5  mm mgso4 , 50  m cacl2 , and 50  m arsenazo iii , or 200  m lumenal pyranine , or 1  m oxonol vi . the reaction was started at 11  c by the addition of 0.2  mm atp and followed by differential absorption spectrometry . b charge measurements on native sr caatpase ( serca1 ) adsorbed on a solid supported membrane ( ssm ) . the current transients were obtained after rapid delivery of 100  m atp to atpase preincubated with 10  m free ca and 100  mm kcl , at ph  7 ( solid line ) or ph  7.8 ( dotted line ) . derived from yu et al . ( 2012 ) 
 a atp - dependent ca uptake , h countertransport , and development of transmembrane electrical potential in reconstituted serca1 proteoliposomes . proteoliposomes prepared at ph  7.0 were diluted ( 5.0  g protein / ml ) in a medium ( ph  7.0 ) containing 10  mm pipes , 100  mm k2so4 , 5  mm mgso4 , 50  m cacl2 , and 50  m arsenazo iii , or 200  m lumenal pyranine , or 1  m oxonol vi . the reaction was started at 11  c by the addition of 0.2  mm atp and followed by differential absorption spectrometry . b charge measurements on native sr caatpase ( serca1 ) adsorbed on a solid supported membrane ( ssm ) . the current transients were obtained after rapid delivery of 100  m atp to atpase preincubated with 10  m free ca and 100  mm kcl , at ph  7 ( solid line ) or ph  7.8 ( dotted line ) . derived from yu et al . ( 2012 ) further evidence for ca / h exchange is provided by measurements of charge transfer upon addition of ca or atp to microsomal vesicles adsorbed on a solid supported membrane ( ssm ) ( tadini - buoninsegni et al . 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 ca , a current transient is observed due to vectorial translocation and dissociation of bound ca in the direction of the ssm electrode after phosphoenzyme formation by utilization of atp ( tadini - buoninsegni et al . 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 . 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 / ca exchange ( i.e. alkaline ph ) , vectorial translocation of bound ca 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 ca sites , in order to obtain ca 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 . cation / h exchange at the transport sites following phosphoenzyme formation occurs in ca atpases , but does not occur in copper atpases ( lewis et al . 2012 ) . the importance of ca / h exchange in determining release of bound ca from the phosphoenzyme can be also demonstrated in steady state experiments . it is shown in fig .  4 that the maximal levels of accumulated ca 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 , ca is less likely to dissociate from the phosphoenzyme . on the other hand , while ca translocation is reduced , steady state atpase activity is increased as the ph is raised , and continues after maximal levels of ca uptake are reached . it is apparent that alkaline ph reduces ca / h exchange and dissociation of bound ca , whereby the phosphoenyme bypasses the ca release step and proceeds to hydrolytic cleavage of pi . therefore , reduction of the ca / atp transport ratio can be produced either by a high ca concentration or a low h concentration in the lumen of the vesicles.fig . 4maximal levels of ca uptake and rates of atpase activity in the absence of oxalate as a function of ph . experiments performed as in fig .  2b , except for ph regulation with 50  mm mes or hepes buffer . the reaction was started by addition of 1  mm atp , at 25  c . original data maximal levels of ca uptake and rates of atpase activity in the absence of oxalate as a function of ph . experiments performed as in fig .  2b , except for ph regulation with 50  mm mes or hepes buffer . the reaction was started by addition of 1  mm atp , at 25  c . original data 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 ca / h exchange and a pathway for slippage of the ca 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 ca , yielding e1    2ca . e1 
 ~  p    2ca , followed by release of adp and utilization of the phosphorylation potential to change vectorial orientation and affinity of the ca sites . bound ca 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 ca sites to the cytosolic medium . the dotted lines in scheme 1 indicate that if lumenal ca is higher than its dissociation constant , or lumenal h is too low to sustain exchange , ca release and formation of nh    e2 are prevented . interference with completion of the atpase cycle would then cause reversal to e1 
 ~  p    2ca ( 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.fig . 5 
 a equilibrium levels of phosphoenzyme obtained through utilization of pi by serca1 at acid or alkaline ph , in the absence or presence of ca . reaction medium : 50  mm mes ( ph  6.0 ) or hepes ( ph  7.5 ) , 20  % me2so4 , 10  mm mgcl2 , 100  mm kcl , 2  mm egta or 1  mm cacl2 ( in the absence of egta ) , and 50  g protein / ml . the samples were acid quenched after 2  min incubation at 30  c , and processed by electrophoresis for determination of radioactive atpase protein . b ca concentration and ph dependence of phosphoryl transfer from phosphoenzyme to adp , to yield atp . phosphoenzyme was obtained by incubating 3.0  mg of protein / ml in 60  mm tris - maleate , ph  6.3 , 4  mm [ p]pi , 20  mm mgc12 , 0.5  mm egta . following a 2  min incubation at 30  c , the reaction mixture was diluted 10-fold with 60  mm tris - maleate ( ph  8.0 ) , 1  mm adp , cac12 and egta to yield free ca as indicated . the samples were acid quenched after 5  min incubation and processed for determination of atp . derived from lewis et al . ( 2012 ) and de meis and inesi ( 1982 ) 
 a equilibrium levels of phosphoenzyme obtained through utilization of pi by serca1 at acid or alkaline ph , in the absence or presence of ca . reaction medium : 50  mm mes ( ph  6.0 ) or hepes ( ph  7.5 ) , 20  % me2so4 , 10  mm mgcl2 , 100  mm kcl , 2  mm egta or 1  mm cacl2 ( in the absence of egta ) , and 50  g protein / ml . the samples were acid quenched after 2  min incubation at 30  c , and processed by electrophoresis for determination of radioactive atpase protein . b ca concentration and ph dependence of phosphoryl transfer from phosphoenzyme to adp , to yield atp . phosphoenzyme was obtained by incubating 3.0  mg of protein / ml in 60  mm tris - maleate , ph  6.3 , 4  mm [ p]pi , 20  mm mgc12 , 0.5  mm egta . following a 2  min incubation at 30  c , the reaction mixture was diluted 10-fold with 60  mm tris - maleate ( ph  8.0 ) , 1  mm adp , cac12 and egta to yield free ca as indicated . the samples were acid quenched after 5  min incubation and processed for determination of atp . derived from lewis et al . ( 2012 ) and de meis and inesi ( 1982 ) 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 ) . 5a that this reaction ( i.e. , reverse reaction of phosphoenzyme hydrolytic cleavage ) is enhanced by acid ph , and is inhibited by alkaline ph and ca . this indicates a requirement for proton occupancy of acidic residues involved in ca 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 ca , in order to replace protons with ca on the low affinity binding sites ( i.e. transition of nh    e2-p to e2-p    2ca and adp    e1  ~  p    2ca ) . 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 ; mller et al . comparison of these structures reveals rearrangements of transmembrane helices upon ca binding , phosphoenzyme formation , occlusion and then dissociation of bound ca , 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 ca binding domains , including the roles of critical amino acids in substrate binding , catalytic reactions , and ca 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 ca , following ca / 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 . 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 ca atpase ( serca1 ) of ( at least human and rabbit ) fast twitch skeletal muscle , and produces uncoupling of atp utilization and ca transport , with a consequent thermogenic effect ( mall et al . 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 ca 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 ca binding affinity ( koss and kranias 1996 ; maclennan and kranias 2003 ; toyoshima et al . 2003 ) and/or a slower e1 to 2ca    e1 transition ( cantilina et al . 1993 ) . this results in a higher ca concentration requirement for ca transport activation ( fig . . following stimulation the cytosolic ca concentration rises from 0.04  m to 0.6  m , and then returns to the 0.04  m resting level within 0.6  s. b rates of ca uptake by cardiac sarcoplasmic reticulum vesicles as a function of free ca concentration . before the measurements , the vesicles were pre - incubated with either a control buffer ( filled circle ) , or with a monoclonal antibody neutralizing phospholamban ( filled triangele ) . note how neutralization of phospholamban decreases the ca concentration required for activation of the transport atpase . note also how the cytosolic ca concentrations observed at the low and high ends of the ca signal , correspond to ca levels indufficient or suitable to yield serca activation . derived from prasad and inesi ( 2012 ) and cantilina et al . ( 1993)scheme 1diagram outlining the sequential reactions on a serca catalytic and transport cycle as explained in the text above . the solid lines indicate the optimal pathway of a well coupled atp utilization and net ca2 + . the dotted lines indicate a short cut of the enyme cycle , whereby atp utilization is uncoupled from net ca2 + transport , as explained in the text above 
 a ca signaling in cultured cardiac myocytes subjected to field stimulation . following stimulation the cytosolic ca concentration rises from 0.04  m to 0.6  m , and then returns to the 0.04  m resting level within 0.6  s. b rates of ca uptake by cardiac sarcoplasmic reticulum vesicles as a function of free ca concentration . before the measurements , the vesicles were pre - incubated with either a control buffer ( filled circle ) , or with a monoclonal antibody neutralizing phospholamban ( filled triangele ) . note how neutralization of phospholamban decreases the ca concentration required for activation of the transport atpase . note also how the cytosolic ca concentrations observed at the low and high ends of the ca signal , correspond to ca levels indufficient or suitable to yield serca activation . derived from prasad and inesi ( 2012 ) and cantilina et al . ( 1993 ) diagram outlining the sequential reactions on a serca catalytic and transport cycle as explained in the text above . the solid lines indicate the optimal pathway of a well coupled atp utilization and net ca2 + . the dotted lines indicate a short cut of the enyme cycle , whereby atp utilization is uncoupled from net ca2 + transport , as explained in the text above 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 ) , and also indicated by nmr ( buffy et al . 2006 ) and cross - linking experiments ( sahoo et al . this is a critical position , since structural studies demonstrate that the helices delimiting this groove undergo displacements affecting ca binding , ca 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 e12ca state . it is worth considering that a slight shift of the ca concentration required for atp activation would affect the rates of ca transport at low levels of cytoplasmic ca , and therefore the efficiency of twitch relaxation . however , it is not likely that the km ( equilibrium constant ) of the ca 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 . considering the possibility of ca pump uncoupling , an important question is whether uncoupling of serca1 interferes with reduction of cytosolic ca below the level allowing contractile relaxation of muscle fibers . the evidence presented above indicates that the ca pump is perfectly coupled when the sr lumen cain is low , even if cytosolic caout 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 ca does not reach a concentration higher than its dissociation from e2-p    2ca , as shown in experiments performed with rabbit native sr vesicles ( fig .  2 ) . on the other hand , when cytosolic ca is reduced to a level producing contractile relaxation , such a ca 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 mg bound e1 state ( toyoshima et al . slippage of the pump would occur if lumenal ca were to become higher than its dissociation constant from e2-p    2ca , and cytosolic ca were to remain above the level required for full atpase activation . this may occur upon prolonged muscle activity , if cytosolic ca is maintained relatively high by multiple action potentials and ca flux through plasma membrane voltage sensitive channels , as expected in shivering thermogenesis . alternatively , a rise of intracellular ph above 7.0 , may affect intracellular ca signaling , as recently reported for g protein signaling ( isom et al . , 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 ca rise to high levels , while cytosolic ca 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 .
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 .