sorcin ( soluble resistance - related calcium binding protein ) is a 21.6  kda protein identified in the cytosol of multidrug resistant cells   that belongs to the penta - ef - hand ( pef ) family , a small group of regulatory calcium binding proteins comprising calpain , alg-2 , grancalcin , peflin and pef1 [ 38 ] .
it has a two - domain architecture , characterized by a flexible and hydrophobic gly / pro - rich n - terminal domain and a c - terminal calcium binding domain containing the five ef - hand motifs ( fig .  1 ) , and dimerizes through the unpaired ef5 hand .
like the other pef proteins , sorcin undergoes a ca - dependent activation that promotes translocation to membranes where interaction with several molecular targets occurs . in turn
, these features render sorcin an effective participant in a number of ca - mediated processes .
sorcin activation is induced by ca binding to the two functionally relevant ef3 and ef2 motifs , that are not paired structurally as in most ef - hand proteins , but are connected by the long and rigid d helix ( fig .  1 ) .
an essential step of sorcin activation therefore consists in the transfer of information concerning ca binding from the site with the highest affinity for the metal , ef3 , through the d helix to ef2 , and from there to the rest of the molecule .
the ensuing conformational change is believed to loosen the hydrophobic and hydrophilic interactions that bring the n- and c - terminal domains together .
this renders both domains available for target protein recognition , in particular the d helix residues .
it follows that the ef3-d helix - ef2 region should be considered as a tightly coupled functional unit .
sorcin is expressed in most human tissues including the heart where ca - bound sorcin interacts with and regulates several ionic channels , such as the l - type voltage - dependent channel , the ryanodine receptor , ryr2 , and the sarcoplasmic reticulum ca - atpase , serca2a , and thus plays an important role in the regulation of the excitation 
discordant effects of sorcin overexpression on cardiac function have been reported . according to seidler et al .  
cardiac - specific overexpression of sorcin in rabbit myocytes and transgenic mice leads to a significant reduction in contractility , while suarez et al .   and frank et al
.   reported that transfection of rat or mouse heart or isolated cardiac cells with sorcin - expressing vectors significantly enhanced cardiac function .
the overexpression of sorcin in cardiomyocytes has also been associated with increased activity of the na  ca exchanger , ncx .
the mammalian ncx family includes three genes ( ncx1 , ncx2 and ncx3 ) with very similar functional properties .
ncx1 , the main isoform expressed in the heart , catalyzes the electrogenic exchange of ca and na across the plasma membrane in both the ca influx and ca efflux mode and is one of the crucial regulators of ca homeostasis within cardiomyocytes and of cardiac contractility .
ncx1 consists of nine transmembrane helices with an extracellular n - terminus and a cytosolic c - terminus that is organized in four domains .
there are two adjacent homologous ca - binding domains , cbd1 and cbd2 , arranged in an antiparallel fashion , that are connected via a regulatory catenin - like domain ( cld ) to the membrane part of the exchanger ( fig .  2 ) .
a rise in cytosolic na stimulates rapidly and then inactivates the exchanger ; in contrast , cytosolic ca activates the exchanger and relieves the na - dependent inactivation [ 2528 ] .
regulation by cytosolic na and ca involves sites that do not participate directly in the ion translocation process .
thus , an amphipathic sequence , the xip region ( exchanger inhibitory peptide ) , takes part in regulation by na , whereas cbd1 and cbd2 are responsible for the ca - dependent activation of the exchanger .
both cbd1 and cbd2 have an ig - like fold with the ca - binding sites in the distal loops .
they contain also an unstructured loop , the fg loop , which in cbd2 is characterized by the presence of a tissue - specific , alternatively spliced region ( fig .  2 ) .
despite the structural similarity , cbd1 binds four ca with high affinity ( kd  =  0.3  m ) , whereas cbd2 binds only two metal ions with lower affinity ( kd  =  5  m ) .
in cbd1 , the four ca ions are brought into close proximity by a complex arrangement of the coordinating residues .
thus , aspartate and glutamate carboxylate oxygens provide bidentate ligands that coordinate two ( asp500 ) or three ( glu451 ) ca simultaneously . in cbd2 ,
the two ca - binding sites are only 5.4   apart , such that the two metal ions are coordinated by two carboxylate oxygens of a single aspartate side chain ( asp578 ) .
mutations of acidic ca - binding amino acid residues in cbd1 decrease affinity for ca , as expected , but do not eliminate ca - dependent regulation .
in contrast , mutations e516l , d578v and e648l in the cbd2 ca - binding sites completely remove ca regulation , placing the exchanger in a constitutionally active state . in accord with these observations ,
the alternatively spliced ncx1 kidney isoform with an arginine at position 578 and the brain isoform carrying a d578r mutation are not regulated by ca 
 . taken together these data point to cbd1 and cbd2 as major determinants of the ncx1 functional regulation .
several experimental data point to an effect of sorcin on ncx function in the heart , indicative of a possible direct interaction between the two proteins .
in particular , ncx activity is increased in sorcin - overexpressing cardiomyocytes , while a low ncx activity has been observed in a rabbit model of left ventricular dysfunction ( lvd ) with a reduced sorcin expression level . in the present work
, the study of the sorcin - ncx1 system was investigated by parallel experiments on the isolated components and on cardiomyocytes .
overlay assay and surface plasmon resonance ( spr ) experiments allowed us to establish the occurrence of a direct interaction between sorcin and the two ncx1 calcium binding domains and to characterize its kinetic and equilibrium parameters , while measurements of the ncx current in cardiomyocytes as a function of voltage furnished information on the functional effect of the interaction .
further , the surface of the sorcin molecule involved in complex formation was determined by means of previously characterized sorcin variants carrying mutations at sites involved in ca binding or information transfer .
the effects of the mutations on the interaction parameters were assessed in surface plasmon resonance experiments , while those on function were determined by means of ncx current measurements .
specifically , the c - terminal , sorcin ca - binding domain ( scbd ) was employed to establish whether this domain is the interacting one ; the e124a mutant was used to gain information on the ca - dependence of the interaction since this variant is unable to bind ca at physiological concentrations due to substitution of the bidentate ca ligand at the ef3 hand ; the w99 g and w105 g variants were utilized as selective probes of the local structural perturbations introduced by substitution of the two tryptophan residues located at the beginning and end of the d helix .
the results obtained point to the ca - dependent interaction between sorcin and ncx1 as the basis for the activation of the transporter . in sorcin , the interacting surface can be mapped on the ca - binding c - terminal domain in the region of the d helix comprising w99 . in ncx1 ,
intriguingly , the rates of complex formation / dissociation are fast and could permit a beat - to - beat interaction .
wild type human and chinese hamster sorcin , scbd ( sorcin calcium binding domain ) and sorcin mutants were cloned , expressed and purified as described previously .
the cdna of mus musculus ncx1 ( cdna clone mgc:90693 image:30532156 ) was used as template in pcr experiments in order to clone the nucleotide regions 11921619 , encoding for cbd1 ( calcium binding domain 1 ) , corresponding to amino acids 397539 , and 15812137 , encoding for cbd2 ( calcium binding domain 2 ) , corresponding to amino acids 527712 .
the pcr products , digested with ndei and hindiii restriction enzymes , were cloned in the pet22 vector .
protein expression was induced by 1  mm iptg for 5  h at 37  c .
after sonication ( 1  min at 14  hz / ml cell solution ) , the lysates were centrifuged at 15,000  rpm for 20  min : cbd1 was recovered in the supernatant , whereas cbd2 remained in the insoluble fraction .
cbd1 purification was carried out by 30% ( w / v ) ammonium sulphate precipitation at room temperature for 1  h , while cbd2 purification required protein extraction by treatment with 0.3% n - lauryl - sarcosine in 50  mm caps buffer ph 11.0 .
an anion exchanger column mono - q ( ge healthcare ) was used to improve purification of both ncx1 domains .
 hcl buffer at ph 7.5 , were loaded onto the column and were recovered upon application of a linear gradient of nacl between 0 and 0.5  m. based on sds page electrophoresis , the degree of enrichment was   80% for cbd1 and   70% for cbd2 . in the spr experiments
the concentration of cbd1 and cbd2 was calculated from the intensity of the respective bands in sds gels ( supplementary fig .
wt sorcin and scbd were subjected to electrophoresis on a 15% polyacrylamide gel under denaturing conditions   and transferred to polyvinyl difluoride membranes ( pvdf ) in transfer buffer [ 25  mm tris 
hcl , 192  mm glycine , and 20% methanol ( ph 8.3 ) ] at 100  ma for 45  min .
the pvdf membranes were incubated at room temperature separately with cbd1 and cbd2 ( 20  g / ml ) in 1% gelatin in tbst buffer [ 20  mm tris  hcl , 0.5  m nacl , and 0.05% tween 20 ( ph 7.5 ) ] containing either 50  m cacl2 ( total concentration ) or 1  mm egta to assess the calcium dependence of the interaction .
subsequently , the membranes were incubated in 1% gelatin in tbst buffer with a mouse anti - ncx1 monoclonal antibody ( antigen : purified canine cardiac ncx ; biocompare ) .
the antibody was used at 1:3000 dilution , a condition where it recognizes equally well cbd1 and cbd2 and does not recognize other proteins like albumin and gelatin , as established in preliminary experiments .
the blots were developed by incubation for 45  min with alkaline phosphatase conjugated monoclonal anti - mouse igg in 1% gelatin in tbst .
control experiments ruled out the existence of cross reactivity between sorcin or scbd and the anti - ncx monoclonal antibody .
surface plasmon resonance ( spr ) experiments were carried out using a biacore x system ( biacore ab , uppsala , sweden ) .
the sensor chip ( cm5 , biacore ab ) was activated chemically by a 35  l injection of a 1:1 mixture of n - ethyl - n-(3-(dimethylaminopropyl ) carbodiimide ( 200  mm ) and n - hydroxysuccinimide ( 50  mm ) at a flow rate of 5  l / min . in one set of experiments ,
the reaction was carried out in 20  mm sodium acetate at ph 6.0 ; the remaining ester groups were blocked by injecting 1  m ethanolamine hydrochloride ( 35  l ) .
this procedure ensures immobilization of sorcin principally via the n - terminus . as a control ,
the interaction of immobilized sorcin with the cbd1 and cbd2 domains of ncx1 was detected through mass concentration - dependent changes in the refractive index on the sensor chip surface expressed as resonance units ( ru ) . the increase in ru relative to baseline indicates complex formation ; the plateau region represents the steady - state phase of the interaction , whereas the decrease in ru represents dissociation of the ncx1 calcium binding domains from immobilized sorcin after injection of buffer .
a response change of 1000  ru typically corresponds to a change in the protein concentration on the sensor chip of 1  ng / mm 
 . the experiments were carried out at 25  c in 10  mm hepes ( ph 7.4 ) , 150  mm nacl , and 0.005% surfactant p-20 .
the concentration of cbd1 and cbd2 ranged between 500  nm and 22  m at a constant calcium concentration . in a second set of experiments ,
cbd1 was immobilized on the activated cm5 chip at ph 4.5 using the procedure described above .
the interaction with wt sorcin or sorcin mutants ( e124a , w99 g , w105 g and scbd ) at a concentration of 1  m was measured in the presence of 20 and 50  m ca .
scatchard analysis of the dependence of the spr signal at steady state ( req ) on the concentration of cbd1 and cbd2 was performed to determine the equilibrium dissociation constant .
the amine coupling kit , the p-20 surfactant , and the cm-5 sensor chip were purchased from biacore ab and all the other reagents were high - purity grade .
negative controls for the spr experiments were obtained by transformation of e. coli bl21(de3 ) with pet22 , induction with 1  mm iptg for 5  h at 37  c , and subsequent sonication and centrifugation ; the supernatants were treated as for the cbd1 and cbd2 preparations , but the last purification step by anion exchanger chromatography was omitted ( supplementary fig .
procedures were undertaken in accordance with the united kingdom animals ( scientific procedures ) act 1986 and conform to the guide for the care and use of laboratory animals published by the us national institutes of health ( nih publication no .
new zealand white male rabbits were given an intravenous injection of 500 u heparin together with an overdose of sodium pentobarbitone ( 100  mg / kg ) and their hearts were removed .
isolated hearts were perfused retrogradely ( 25  ml min , 37  c ) with a nominally ca - free krebs 
henseleit solution containing 0.6  mg ml collagenase ( type 1 , worthington chemical co ) and 0.1  mg ml protease ( type xiv , sigma chemical co ) for 68  min .
after isolation of the left ventricular free wall , the epicardial layers of the free wall were isolated from the remaining tissue by dissecting a 11.5  mm layer from the epicardial surface .
the layers of tissue were incubated separately for 5  min in enzyme solution containing 80  mol / l cacl2 and 4% bovine serum albumin ( bsa , fraction v , sigma ) .
isolated cardiomyocytes were superfused with a hepes - based tyrode 's solution at 3637  c in a chamber mounted on the stage of an inverted microscope .
voltage clamp was achieved a using an axoclamp 2a amplifier ( axon instruments , foster city , ca , usa ) in discontinuous ( switch clamp ) mode .
pipette resistance was 12  m. the tyrode 's solution superfusing isolated cells contained ( mmol / l ) : nacl ( 140 ) , kcl ( 4 ) , hepes ( 5 ) , mgcl2 ( 1 ) , cacl2 ( 1.8 ) , and glucose ( 11.1 ) , ph 7.4 with naoh .
the perfusate also contained strophanthidin ( 0.01  mmol / l ) and nifedipine ( 0.01 
mmol / l , to block k currents ) and niflumic acid ( 0.1  mmol / l , to block ca - activated cl currents .
the pipette solution contained ( mmol / l ) : cscl ( 45 ) , egta / caegta ( cs 100 , egta 50 , ca 25 ) , hepes ( 20 ) , mgcl2 ( 11 , calculated free mg    1.2 
this pipette solution was designed to buffer [ ca]i to   250  nmol / l ( confirmed by separate [ ca ] measurements ) . after achieving the whole - cell configuration , a period of 510  min was allowed for dialysis of the pipette solution into the cell .
currents were then measured in response to a 3  s ramp from   120  mv to +  80  mv from the holding potential of   80  mv .
an ascending ramp was chosen since this has been shown to cause less perturbation of sub - sarcolemmal [ ca ] than a descending ramp , and the resulting currents are closer to those obtained using a voltage step protocol .
the ramp protocol was performed at 0.1  hz , when steady - state currents were achieved ; data from 12 ramps were recorded for subsequent averaging .
the protocol was repeated in the presence of 5  mmol / l ni to obtain the background current , and this was subtracted to obtain the current attributable to ncx ( see fig . 
sorcin or the mutant proteins were added to the intracellular pipette solution for dialysis into the cell at a concentration of 3  m using a protocol similar to previous work , designed to supplement endogenous intracellular sorcin by comparable amounts of exogenous protein .
allowing sorcin to dialyse into the cell for 10 to 20  min had no additional effects ( data not shown ) . preliminary experiments using 1  m sorcin had no significant effect on the ncx current ( see supplementary fig .
as previously described , a partial rabbit sorcin sequence was amplified using primers based on the known human sorcin sequence by reverse transcription of rna from a chinchilla bastard rabbit .
sense and antisense oligonucleotides were chosen to target the 19 nucleotide segment complementary to the rabbit sorcin mrna sequence gcaagaucaccuucgauga and ligated downstream of a u6 promoter into vector psiren - dnr ( bd biosciences clontec , palo alto , usa ) .
six different sirna sequences were cloned into psiren and verified for their effectiveness in a rabbit tumor cell line vx2 .
recombination with the remaining adenoviral genome using padeno - x vector was carried out according to the manufacturer 's instructions ( bd adeno - x expression system , bd , biosciences clontech , palo alto , usa ) . infection of adult ventricular cardiomyocytes with the adrnai virus ( 100 moi ) resulted in downregulation of native sorcin expression to 27    7% of control value after 36  h of quiescent culture .
example blots demonstrating of downregulation of sorcin rna and protein are shown in the online supplement ( supplementary fig .
 sorcin is a penta - ef - hand protein that interacts with intracellular target proteins after ca2 + binding . 
 the sarcolemmal na+/ca2 + exchanger ( ncx1 ) may be an important sorcin target in cardiac muscle . in this study , rnai knockdown of sorcin , 
 purified sorcin or sorcin variants was employed in parallel measurements of : ( i ) ncx activity in isolated rabbit cardiomyocytes using electrophysiological techniques and ( ii ) sorcin binding to the ncx1 calcium binding domains ( cbd1 and ( iii ) using surface plasmon resonance and gel overlay techniques . 
 sorcin is activated by ca2 + binding to the ef3 and ef2 regions , which are connected by the d helix . to investigate the importance of this region in the interaction with ncx1 , 
 three variants were examined : w105 
 g and w99 g , mutated respectively near ef3 and ef2 , and e124a that does not bind ca2 + due to a mutation at ef3 . 
 downregulation of sorcin decreased and supplementation with wt sorcin ( 3  m ) increased ncx activity in isolated cardiomyocytes . 
 the relative stimulatory effects of the sorcin variants were : w105 g  >  wt sorcin  >  sorcin calcium binding domain ( scbd )  >  w99 g  >  e124a . 
 sorcin binding to both cbd1 and 2 was observed . in the presence of 50  m ca2 + , 
 the interaction with cbd1 followed the order w105 
 g  >  scbd  >  wt sorcin  >  w99 g  >  e124a . in sorcin 
 , the interacting surface can be mapped on the c - terminal ca2 + -binding domain in the d helix region comprising w99 . 
 the fast association / dissociation rates that characterize the interaction of sorcin with cbd1 and 2 may permit complex formation / dissociation during an excitation / contraction cycle . 
Introduction
Materials and methods