Journal of Cell Science h -emnsitrcin eitn ermrasml r novdi y hne lsr,ietfigacuilrl o this for role crucial a identifying closure, channel RyR Ca in intracellular involved of regulation are dynamic assembly the tetramer in association mediating structural interactions N-terminus the hra h er rmrl xrse y2 h vital The results conditions RyR2. regulation pathological channel Ca defective abnormal in expresses in acquired highlighted or is primarily inherited where RyRs heart of muscle, skeletal importance In in the isoform 2007). predominant Lai, whereas the and is the (Zissimopoulos RyR1 triggering heartbeat mammals, for each also of and activation contraction muscle skeletal initiating h nerlmmrn yndn eetr(y)gvrsthe governs Ca (RyR) (SR) receptor reticulum ryanodine sarcoplasmic membrane integral The Introduction of dysregulation words: in Key results RyR2 within interactions in enabling inter-subunit occurs oligomerize, N-terminal formation to of endo/ bond ability Disruption the disulfide intrinsic RyR1. Ca from endogenous the in diastolic by at efflux possesses not activation mediated calcium notably fragment channel tetramerization but N-terminal mediating N-terminus RyR2, RyR2 gel subunits Interestingly, native and a is identical crosslinking formation. N-terminus that chemical RyR2 tetramer four analysis, the demonstrate co-immunoprecipitation apparent that of screens, indicating collectively two-hybrid evidence composed assays yeast of of lines channel filtration several combination present A ion We self-association. cells. of an non-excitable capable and is excitable of (RyR) reticulum sarcoplasmic receptor ryanodine The Summary 10.1242/jcs.133538 doi: 5042–5051 126, ß Science Cell of Journal 2013 July 30 Accepted ( correspondence for *Author 2 1 Zissimopoulos Spyros receptors of ryanodine function cardiac the regulates oligomerization N-terminus 5042 soito ewe -emnladcnrldmisrslsin results domains central and N-terminal the between and of disruption association Matsuzaki mutation-induced that Yano, proposed have Ikemoto, colleagues Importantly, RyR example, in 2007). For implicated Lai, been pathophysiology. of inter-subunit have and closing interactions and inter-domain and (Zissimopoulos Intra- defective opening pore the 2003). channel regulate al., the to et believed are Masumiya N-terminal interactions 2004; (George large al., fragments native assays RyR2 et transmembrane complementation C-terminal of The and protein studies N- by the overlapping and 1996). proteolysis between 1993) al., partial with et al., (Chen by RyR1 associated indicated et as is George Wang assembly, 1997b; portion al., 2004; et cytoplasmic Bhat al., 1997a; al., et et (Bhat pore conducting n skont ermrz n omacntttvl pnCa open constitutively a form C-terminus and tetramerize RyR to known is The and 2008). (Fleischer, ( channels of 2009; permeable subunits identical Lai, four and comprise , RyRs (Blayney 2007). failure Lai, and heart George and arrhythmias cardiac lnJ Williams J. Alan ihr Stewart Richard , eatet fBooia cecsadPamclg,Vnebl nvriySho fMdcn,Nsvle N322 USA 37232, TN Nashville, Medicine, of UK School 4XN, University CF14 Vanderbilt Cardiff Pharmacology, Medicine, and of Sciences School Biological University of Cardiff Departments Institute, Research Heart Wales 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. 6 D htcmiet omhg-odcac,cation- high-conductance, form to combine that kDa 560 0 ftepoen otistemmrn-pnigdomains membrane-spanning the contains protein) the of 10% ada yndn eetr -emns acu ees hne,Oligomerization channel, release Calcium N-terminus, receptor, ryanodine Cardiac 2+ adigadlast ermsua disorders, neuromuscular to leads and handling 1 1 oc .Jeyakumar H. Loice , [email protected] n .AtoyLai Anthony F. and 1, ,Cdi Viero Cedric *, 2+ 2+ ocnrtosfo yndn idn n igecanlmaueet.Orfnig ugs that suggest findings Our measurements. channel single and binding ryanodine from concentrations ees hti seta for essential is that release ) 1 2 1 inyFleischer Sidney , oiaSeidel Monika , 2+ - 2+ 1 ea Cumbes Bevan , release. bomlRRCa RyR abnormal nodrt dniywehrayo h y2domain(s) (AD4L; RyR2 RyR2 the of of fragment any N-terminal whether the identify with to associates order tetramers In into assembles N-terminus RyR2 Results 2005). al., et been (Zissimopoulos previously form has abstract results in these of presented Part channel. the of tetramerizatio conformation N-terminus RyR2 presen and channel tetrameric RyR2 occurring interaction RyR1 2010). native al., the et N- recently (Tung within RyR1 map was 1–559) an density cryo-EM of acids axis structure (amino crystal fourfold fragment the terminal central docking on the based proposed, surrounding specifically 2007). that al., it topology et N-terminal Wang located different 2008; very al., a al., et contrast, Serysheva et By 2001; (Baker al., architecture et three-dimensional Liu 2002; RyR the docking the at of region and clamp or corners the modeling within N-terminus GFP-RyR homology the placed of have or Initial analysis, channel. fusions, (EM) the protein microscopy of GST-RyR cryo-electron module important using functionally reports a implying genetic respectively, is CPVT numerous it and that containing MH each with associated clusters catecholaminergic mutations disease major and three and (Ikemoto 2009). al., (CPVT) et Yano (MH) tachycardia 2002; Yamamoto, ventricular hyperthermia polymorphic malignant 2 atrh Mukherjee Saptarshi , ee erpr nteietfcto fanvlinter-subunit novel a of identification the on report we Here, the of one of location the is RyR2 and RyR1 of N-terminus The 2+ 1 ees n sivle nteptoeei of pathogenesis the in involved is and release uihWhite Judith , ewe h -emnldmiswti the within domains N-terminal the between 1 .LwiThomas Lowri N. , ninstabilizationoftheclosed ucinleiec implicating evidence functional t 1 rsCheung Iris , eerhArticle Research 1 , 1 , Journal of Cell Science ie lgmrhdfre opiigB4 n DL This AD4L. and BT4L comprising formed had oligomer mixed a y-TLwsaaye yimnbo sn antibodies using immunoblot by (Ab Myc analyzed against was Myc-BT4L agdB4 ( BT4L tagged pcfclycridoti ihrrdcn 1 MDT or DTT) were mM experiments (10 further reducing H either so mM (1 in and oxidizing out species carried oligomeric specifically an of ihGL N-D,adwal neatdwt h y2C- RyR2 the with interacted (AD4L) weakly fused N-terminus and 1–906 DNA-BD), residues RyR2 GAL4 (BT4L; human with itself with the potently most that associated found 1). see (Fig. we protein entire Notably, AD; RyR2 the spanning overlapping for GAL4 constructs with screen expression AD4L to domain with of system interaction (Y2H) fused protein two-hybrid potential yeast the the RyR2 used we human 1), Fig. of 1–906 residues noe A4DAB uinwt 5 rti;pD noe A4A uinwt V0lreTatgn.Rslsaemeans are Results antigen). T large SV40 with fusion liquid AD Quantitative GAL4 construct. encodes AD4L pTD1 N-terminal protein; RyR2 p53 the with with fusion cells. interaction DNA-BD yeast for GAL4 in system encodes itself Y2H with the interacts in N-terminus tested RyR2 The 1. Fig. ( with immunoprecipitated was (Ab co- lysate antibodies CHAPS- performed anti-HA cell from then HEK293 HA-AD4L we (1–906), solubilized assays. and epitope (co-IP) peptide N-terminus respectively), HA immunoprecipitation AD4L, or RyR2 Myc and the the (BT4L either with cells tagged differentially HEK293 mammalian data BT2 oligomerization. These subunit overlapping RyR2 AD4L. cogent facilitating confer specifically thus to their can self-association, portion binding N-terminal and the that negligible suggest BT6 showed (C-terminus–N- (BT5, fragment) BT8–AD4L constructs ( weaker the domain considerably Quantitative was whereas interaction 1, terminus) Fig. table). the histogram), for N-terminus–N-terminus; inset interaction inset (i.e. strong association 1, very (Fig. a BT4L–AD4L indicated (BT8) assays galactosidase tail terminal n o ntecnrlI akn nioy ihu DTT Without antibody. Ab lacking IP additional control an the in (ambient), not and , oetn h 2 bevtos ec-xrse in co-expressed we observations, Y2H the extend To 400 6 10 3 adwsdtce nteAb the in detected was band ) , Myc 0 D)wsrcvrdol nteAb the in only recovered was kDa) 100 .Iiilosrain eeldtepresence the revealed observations Initial ). 2 O 2 HA odtos i.2sosta Myc- that shows 2 Fig. conditions. ) n h rsneo co-precipitated of presence the and ) Myc imnratv high -immunoreactive HA P niaigthat indicating IP, , 0) Central 10%). ceai iga eitn h eiso ua y2oelpigpoenfragments protein overlapping RyR2 human of series the depicting diagram Schematic HA IP, M b - r h xdzn egn H reagent However, oxidizing protomers. the between the bonding disulfide the suggesting of addition, existence DTT by abolished was species oligomeric sRR -emnlttaesit ooes(a ta. 1989; al., et (Lai well monomers as into of RyR1, was tetramers native use which C-terminal of the RyR2 3-14, dissociation as cause with zwittergent protein- to tested detergent shown non-covalent previously further self- zwitterionic by was linear N-terminal the This that mediated RyR2 interactions. indicating and primarily 2), protein (Fig. dispensable is DTT are association agent bonds reducing disulfide the when of presence tetramers DTT-sensitive into cells. mammalian self-associating in expressed be may is S- domain bonds, , BT4L–AD4L disulfide the inter-subunit 2006). As al., or et (Aracena-Parks intra- S-nitrosylation modification; and glutathionylation RyR1 of types to three all oxidation displayed N-terminus, cysteines it including the cysteines, Indeed, within RyR1 several disulfides. that other to reported been oxidation in has their S-nitrosylated in preventing formation or thus S-glutathionylated bond subunits, disulfide are in subunits participating Possibly, some form. cysteines oligomeric same the into the monomeric the of conversion uui neatos natraieepaaini that causes self-association. the detergent, is its of preventing unfolding powerful thereby CHAPS explanation partial that N-terminus, and/or relatively RyR2 changes co- of alternative suggests as conformational protein An presence 3-14, in which inter- zwittergent interactions. the N-terminal S1), AD4L RyR2 non-covalent, subunit in Fig. disrupts and 3-14 zwittergent material retained BT4L (supplementary was self- BT4L–AD4L between robust interaction whereas prevented 3-14 assays, interaction zwittergent immunoprecipitation that found We the 2003). al., et Stewart 0 D rti togysget htteRR N-terminus RyR2 the that suggests strongly protein kDa 400 oal,B4–DLsl-neato curdi the in occurred self-interaction BT4L–AD4L Notably, y2Ntriu ermrzto 5043 tetramerization N-terminus RyR2 b glcoiaeasy r hw nteist(pVA3 inset the in shown are assays -galactosidase 2 O 2 i o usatal enhance substantially not did , 0 D,fraino the of formation kDa, 100 6 s.e.m. Journal of Cell Science muorcpttdwt Ab with immunoprecipitated tnad ooe n ie ermr r hw ihtearrows. the mass with molecular shown as are serve tetramers to mixed included and also Monomer was standard. samples, IP in processed n h rsneo soitdB4 a nlzdb D-AE(%gel) (6% SDS-PAGE by analyzed Ab was using BT4L immunoblotting associated and of presence H the mM and 1 or DTT mM pre- 10 1–906, without residues or RyR2 with (AD4L) treated HA-tagged and (BT4L) mammalian Myc-tagged in fragment N-terminal RyR2 cells. of Self-association 2. Fig. 5044 Fg AB niaigeitneo ermrcasml of assembly ( tetrameric data a Cumulative a of existence of , indicating cell formation 3A,B) (Fig. HEK293 time-dependent crosslink the observed to expressing glutaraldehyde homogenates used we oligomer, n rti tnad iharneo 040ka(i.3D). (Fig. to kDa oligomer 30–460 the calculated of 358 we range curve, be standard a resulting the with gels From SDS-PAGE standards gradient protein 4–15% used and we oligomer, BT4L the of small disulfide-linked a a that as exists indicate already tetramer. As experiments N-terminus 3B). the these (Fig. of assays, DTT) proportion mM co-IP DTT 10 the by point, abolished with is time but minute point), (0 time minute pre-treatment 0 glutaraldehyde trimer the before at even no (i.e. tetrameric conditions addition ambient the and in component, evident dimer minor is a observed minimal form is band it with Although protein detected. tetramer, bands predominant a The to 3C. corresponds Fig. in presented 00.Hwvr ieecuincrmtgah niae that al., indicated et chromatography (Tung electron exclusion arrangement cryo-EM size tetrameric RyR1 However, a 2010). native suggested the map density onto and docking determined was sulfhydryl topological 1–559) (residues fragment that N-terminal RyR1 suggesting cells. 3D), the (Fig. within endogenously treatment occurs oxidation obtained NEM (a still were NEM after Tetramers to mM sulfhydryls. homogenization free 5 modify cell included expected covalently air during by we reagent) the formed alkylating experiment, than are thiol-reactive, the bonds size disulfide during smaller whether oxidation address slightly To an a kDa. have 400 could to it corresponds therefore SDS-PAGE and that during form, linear mobility equivalent relative the greater retardation than with gel circular run less closed would experience and A should channel. species RyR2 the protein of native tetrameric arrangement the the within from subunits expected four as fashion, circular closed 0 D TLpooes repcieo T pre-treatment. DTT of irrespective protomers, BT4L kDa 100 oeaietepeiesocimtyo h y2N-terminus RyR2 the of stoichiometry precise the examine To nodrt cuaeydtrieteaprn oeua mass molecular apparent the determine accurately to order In uigtecus foreprmns h rsa tutr fa of structure crystal the experiments, our of course the During oimnpeiiainasy rmHK9 ellst co-expressing lysate cell HEK293 from assays Co-immunoprecipitation 6 5ka( kDa 15 ora fCl cec 2 (21) 126 Science Cell of Journal n 5 n 5 ) ossetwt ermrarne na in arranged tetramer a with consistent 4), )floigdnioer nlssare analysis densitometry following 4) HA Myc rmCASslblzdHK9 lysate HEK293 CHAPS-solubilized from ellst,15t ftevolume the of 1/50th lysate, Cell . , 0 D TLpoen We protein. BT4L kDa 100 2 O 2 sidctd DLwas AD4L indicated. as , 0 D band kDa 400 Rf rget eemntrdb muoltuigtodifferent two RyR2 using (Ab immunoblot calpain-cleaved antibodies by N-terminal-specific and monitored were Untreated fragments fragments. proteolytic nioyaantteCtriu (Ab C-terminus the against antibody w diinlbnswudb h ullnt ermrat at tetramer tetramer full-length N-terminus the the be and would are kDa bands that 2200 and additional tetramers kDa into 550 two bands to form two then addition N-terminus bonds, in disulfide its endogenous by and linked 5A,B DTT. covalently RyR2 agent, Fig. reducing in the the SDS-PAGE with observed treated If of were we samples result where what 3, a is lane This disulfide as and N-terminus conditions). endogenous dissociated kDa denaturing its by are 550 linked (tetramers and the not observed and RyR2 are only the kDa but 550 N- bonds, If tetramers its the N-terminus. as and exist the only RyR2 of form, the analysis monomeric If that in evident. , protease exist also is terminus endogenous intact; fragment largely by terminal) is the degradation subunit show generates kDa) digestion, some (550 calpain exogenous RyR2 of however, full-length absence the the in Lanes that 5, follows. and as 3 possibilities, 1, different the between distinguish -emnsi h aieRR rmpgcricS,under H SR, mM cardiac (1 conditions pig oxidizing from or DTT) RyR2 mM native (10 the reducing the of state in oligomeric RyR2 the N-terminus investigated adjacent this therefore favor of We not RyR2, might interaction. proteins folding full-length regulatory accessory the protein and/or domains in disparate the chemical present potentially be that where co-IP, this not However, suggest might tetramerize. Y2H, can strongly oligomerization region the data N-terminus RyR2 gel-filtration discrete from and evidence crosslinking cumulative The disulfide-linked into tetramers assembles N-terminus RyR2 Native aieRR a nuae ihclanpoes ognrt the generate to protease calpain characteristic with incubated was RyR2 Native omto yprfe S-TLwsas eosrtdby demonstrated S3). bands Fig. trimer also material or (supplementary dimer observed was appreciable no protein GST-BT4L with crosslinking, a purified chemical and by monomers conditions,formation into only GST- denaturing dissociate at gel-filtrated band SDS-PAGE will interactions, tetramers of preserved. protein–protein BT4L result disrupt be by a which together will as non-denaturing held proteins interactions However, oligomers under separates protein–protein protein that size non-covalent therefore technique hydrodynamic and a their ml) conditions, (64–68 is We to filtration respectively. fractions monomer, according gel elution and that tetramer GST- later note the 4). and (Fig. to corresponding kDa ml) early 29–669 (46–52 profile, elution the of of fractions areas distinct range two in detected the was BT4L in standards protein 1 MDT n hnteeue rcin,aaye by analyzed fractions, eluted conditions the reducing Ab then under bacteria using and exclusion from, immunoblot DTT) size mM purified by (10 separated ( and GST-BT4L Thus, was S2). in, Fig. material (supplementary assess expressed to protein filtration (GST-BT4L) recombinantly gel RyR2(1–906) human used a the therefore of remained oligomerization We fragment solution. in RyR1 monomer bacterially-expressed purified, the 5 D ad hudb bevduo immunoblot upon observed be should bands kDa 150 ntebsso h eut band(i.5,) ecan we 5A,B), (Fig. obtained results the of basis the On , , 3 D ilb eetd(i.4,) Tetramer 4A,B). (Fig. detected be will kDa 130 5 D -emnland N-terminal kDa 150 , 5 D -emnl(and N-terminal kDa 150 GST eecmae ihgel-filtrated with compared were , , 5 D ol eepce;the expected; be would kDa 150 , D2 5 D ad hudbe should bands kDa 150 1093 n Ab and ). , 0 D C-terminal kDa 400 H300 , , 0 D.We kDa. 600 n ihan with and ) 0 D C- kDa 400 , 3 kDa) 130 2 O 2 ). Journal of Cell Science r ie sma value mean as given are -tymliie rtiswr eaae hog –5 rdetSSPG e oehrwt ihmlclrms akr HMr,Ivtoe) A Invitrogen). (HiMark, markers mass molecular high plotting with by together prepared gel subsequently SDS-PAGE was gradient curve 4–15% standard through calibration separated were Proteins N-ethylmaleimide. ( analysis Densitometry (C) ae n abetadH and (ambient 5 and 1 lanes -emnlfamn.Adto fH of Addition fragment. the N-terminal of tetramerization DTT, disulfide-linked by with abolished was consistent it a but cleavage, detected calpain following persisted N-terminus, the within , epitopes different recognize eepce.Ti swa eosre nFg ABlns2ad6 at and 2 lanes by 5A,B tetramer Fig. H in linked observed and N-terminus we (ambient what covalently is This the expected. are be bonds, that , tetramers samples disulfide where N- as its 4, and endogenous RyR2 lane exist the 5A,B If DTT. Fig. endogenous agent, terminus reducing in the by observed with treated linked we were what not is the are This only that bonds, tetramers disulfide form only terminus monomeric, were N-terminus its the and RyR2 the abolished If is terminus). subunit the kDa calpain yield 550 exogenous low full-length following to large to 6, the a due and that 4 show such probably 2, digestion, of most lanes 5A,B transfer is Fig. electrophoretic visible protein. poor not and/or is band abundance kDa 2200 the bevdthe observed glutaraldeh with incubated Ab was using homogenate immunoblotting Cell and DTT). gels) mM (6% (10 SDS-PAGE dithiothreitol by agent, analyzed reducing and fragment. the points N-terminal (B) time RyR2 with indicated of or the (A) form without oligomeric pre-treated predominant BT4L, the expressing is Tetramer 3. Fig. 0 D lgmrcpoen(i.5,) h adat band The 5A,B). (Fig. protein oligomeric kDa 600 0 D nadto othe to addition in kDa 600 hs w ifrn niois Ab antibodies, different two Thus, , 5 D adsol eosre.I h y2adisN- its and RyR2 the If observed. be should band kDa 150 , , 5 D -emnlfamn (and fragment N-terminal kDa 150 0 D,btntte20 D adi i.5A,B Fig. in band kDa 2200 the not but kDa, 600 2 O 2 respectively). , 6 ...()HK9 el xrsigB4 eehmgnzdi h rsneo 0m T,1 mM 10 DTT, mM 10 of presence the in homogenized were BT4L expressing cells HEK293 (D) s.e.m. n 5 )wscridoto h ad orsodn ottae n ooe n sdt xrs ermrt ooe ai.Data ratio. monomer to tetramer express to used and monomer and tetramer to corresponding bands the on out carried was 4) , 5 D adsol eobserved. be should band kDa 150 2 O , 2 5 D ooe adwould band monomer kDa 150 epciey.Terao why reason The respectively). , 2 O D2 2 i o substantially not did n Ab and , H300 0 D C- kDa 400 , , Rf 5 kDa 150 kDa 600 which , ausvru log versus values pcfcfrteN n -emnlRR rget (Ab fragments RyR1 antibodies C-terminal using and SR, N- muscle the skeletal for rabbit specific from RyR1 native calpain for targeted digestion Alternatively, specifically digestion. linkage. calpain be endogenous disulfide might endogenous RyR2 inter-subunit by disulfide-linked that for cleaved imply RyR2 already primes could are This exogenous disulfide-linked calpain. following are increase subunits RyR2 that native substantially that seems it to Interestingly, digestion. calpain appear not did soitswt tefad fe api laae remains cleavage, calpain region both N-terminus after that Note RyR2 the bonds. and, disulfide pig DTT-sensitive disulfide-linked itself through native tetrameric with with the consistent that associates be suggest results the would of which tetramerization DTT, by , emnsttae omnmrrltv bnac s10.8 is 13.0 abundance relative and monomer to tetramer N- disulfide-linked terminus the cleavage, calpain exogenous following that the enhance ( h y2Ctriu Fg C.Ised Ab Instead, 5C). (Fig. C-terminus RyR2 the the detect to n 5 60ka hc eitdclancevg u a abolished was but cleavage calpain resisted which kDa, 1600 iia api laaeeprmnswr are u for out carried were experiments cleavage calpain Similar M , ) ycnrs,Ab contrast, By 4). r 0 D -emnsand N-terminus kDa 600 n itn h aauigaplnma uv ntshown). (not curve polynomial a using data the fitting and AB hmclcosikn sasfo E23cl homogenate cell HEK293 from assays crosslinking Chemical (A,B) 6 .%frabetadH and ambient for 4.7% Myc , , y2Ntriu ermrzto 5045 tetramerization N-terminus RyR2 lgmrcfrsaeidctdb h arrows. the by indicated are forms Oligomeric . 0 D pce.Dnioer nlssindicated analysis Densitometry species. kDa 600 0 D ad ofrigta tde o contain not does it that confirming band, kDa 600 1093 , 0 D -emnlfamn.These fragment. C-terminal kDa 400 seii o y2Ctriu)failed C-terminus) RyR2 for (specific , 60kaCtriu tetramers C-terminus kDa 1600 2 O 2 b odtos respectively conditions, mratehnlo mM 5 or -mercaptoethanol 1093 eetdabn at band a detected 6 2142 d for yde 4.0% or Journal of Cell Science loo eyrgns 10ka,BA(6ka n abncanhydrase carbonic and kDa) (66 kDa). BSA (29 kDa), kDa), (150 (443 dehydrogenase apoferritin alcohol kDa), (669 thyroglobulin using airto uv euinvlmsi lpotdaantlog against plotted Gel-filtration ml (C) in 1–906). volumes (elution residues curve (RyR2 calibration GST-BT4L purified of filtration ermr(i.5F). (Fig. tetramer a of appearance netgtdteefcsta h TLfamn xrso the on we exerts significance, fragment BT4L functional the putative that effects its RyR2 the the assess investigated of could regulation To but functional channel. to role, contribute structural potentially a also confers tetramerization activity channel N-terminus enhances dissociation N-terminus RyR2 H but DTT-treated), (ambient, monomer a RyR1 as found The 5D,E). (Fig. Ab Ab fragment. using N-terminal analysis RyR2 blot purified Western of (A,B) filtration Gel 4. Fig. ooe,irsetv ftepeec fDTo H or DTT of presence the of irrespective monomer, , 5046 5 D -emnlfamn a eetdol sa as only detected was fragment N-terminal kDa 150 H300 n Ab and ora fCl cec 2 (21) 126 Science Cell of Journal 2149 , 60kabn,wihi ossetwt a with consistent is which band, kDa 1600 epciey.Ulk y2 h RyR1 the RyR2, Unlike respectively). , , 0 D -emnlfamn was fragment C-terminal kDa 400 GST feue rcin olwn gel following fractions eluted of b ayae(0 kDa), (200 -amylase M r a generated was ) 2 O 2 induced 2 O 2 xrse TLddntafc yndn idn oRR at RyR2 to binding ryanodine affect co- not of Ca expression, did presence The BT4L immunoblotting. protein by expressed verified RyR2 also was equivalent which indicating respectively, ugsigta y2canlatvto yterecombinant the by Ca activation low in channel occurs BT4L RyR2 that suggesting TLt h i hme eutdi infcn nraeo open of increase significant ( a in resulted probability chamber cis the to BT4L prxmtn h isoi Ca diastolic the approximating 37.9 of caffeine) mM 10 eeldamxmmbnigvle(n100 (in value binding alone, maximum RyR2 or a RyR2+BT4L, revealed microsomes co-expressed HEK293 either on contained DTT) that mM (2 conditions reducing under ihntv y2adte10kaclancevdN-terminal [ calpain-cleaved Initial kDa S4). Fig. 150 material interacts the (supplementary region polypeptide and fragment of GST-BT4L assays, RyR2 N-terminal purified pull-down native interaction GST the with endogenous Using that the channel. observed the RyR2 we facilitating native with the thus within BT4L bonds, N-terminus, disulfide exogenous covalent RyR2 within was the interactions the protein–protein reduce DTT non-covalent RyR2 to the inter- function. only assays the leaving channel the N-terminal within altering in endogenous included and exist interactions disrupting N- to subunit for presumed thereby compete could sites oligomer, fragment binding BT4L exogenous terminal the reasoning Our that channels. RyR2 was recombinant and native of activity ssoni i.7,RR hne ciiywseteeylow extremely was Ca to activity cytosolic channel nM RyR2 RyR2 100 7A, at full-length Fig. channel. in oligomeric the shown the As with within potentially dissociation BT4L action N-terminus BT4L promote of of interaction mechanism concentrations kinetic involving protein BT4L the nM) a and could (10 low applied, channels of relatively some the probability to of due open be delay The mM variable delay. the variable (+10 and/or a chamber, following unresponsiveness increased, cis polypeptide channels RyR2 the of BT4L third to addition recombinant DTT) Upon myocytes. nbiga nacdcnetaino TLpoento protein BT4L of concentration enhanced cells, HEK293 in an RyR2 full-length enabling with BT4L co-expressed we and/or bacteria) time. low incubation from the assay yield by binding purification (dictated insufficient and used to GST-BT4L expression due of protein be could concentration This low significance. GST- statistical the the reach although nM not 10 S1), did results Table that material indicated a (supplementary SR concentrations caused cardiac pig BT4L of assays binding aeilFg 5.[ (supplementary S5). RyR2 full-length Fig. recombinant the material with translocates only localization upon co-expression microsomal specifically predominantly upon a BT4L self- to cytosol recombinant channels the N-terminus from the analysis immunoblot that the RyR2 and indicated fractionation disrupt Sub-cellular within and expression. with association interact potentially significant ( nraein increase hne ciiywsmntrda 0 Mfe i Ca cis free nM 100 at cation monitored and was bilayers gradient lipid activity density into channel sucrose incorporated by was isolated CHAPS- centrifugation from cells RyR2 HEK293 recordings. solubilized single-channel in investigated # ooecm hspolmo h o il fioae protein, isolated of yield low the of problem this overcome To h ucinlefcso TLo y2atvt eefurther were activity RyR2 on BT4L of effects functional The 2+ 5 M,tepeec fB4 nue statistically a induced BT4L of presence the nM), 250 § 1 m Fg ) ycnrs,a o Ca low at contrast, By 6). (Fig. M P , o P wfl nrae( increase twofold o yGTB4 a 9.8 was GST-BT4L by 5 .6) ne hs odtos h average the conditions, these Under 0.064). , wfl idn nraea o Ca low at increase binding twofold 3 ]yndn idn sasperformed assays binding H]Ryanodine 2+ 2+ ( conditions. P o 5 6 .0) diino 0n GST- nM 10 of Addition 0.004). P . n 41.0 and 4.3 , 2+ .5 nraoiebinding, ryanodine in 0.05) 6 ocnrto ncardiac in concentration . od( fold 4.4 m reCa free M 2+ n 6 concentrations 5 3 . fmol/mg, 3.7 H]ryanodine channels). 5 2+ and 2+ 2+ , Journal of Cell Science D,Ab (D), sn Ab using BT4L. aaaenraie gis aiu idn otie ntepeec of presence the in (obtained duplicate. binding in maximum 100 least against at normalized performed are each Data experiments separate three of Summary , y2aoeo y2wt TL vrarneo reCa free of range a over BT4L, with RyR2 or alone RyR2 i.6 y2dsly nrae [ increased displays RyR2 6. Fig. H mM 1 or DTT mM 10 without or tetramers. with disulfide-linked pre-treated into were assembles that N-terminus (D–F), RyR2 Native 5. Fig. 0 D -emnlfamnsa ela lgmrcfrsaeidctdb arrows. by indicated are forms oligomeric as well as fragments C-terminal kDa 400 m reCa free M [ H300 3 D2 ]yndn idn saso E23mcooe expressing microsomes HEK293 of assays binding H]ryanodine gis y2Ntriu A,Ab (A), N-terminus RyR2 against gis y1Ntriu E n Ab and (E) N-terminus RyR1 against 2+ n 0m afie n xrse sma value mean as expressed and caffeine) mM 10 and 3 ]yndn idn ntepeec of presence the in binding H]ryanodine H300 2149 gis y2Ntriu B,Ab (B), N-terminus RyR2 against gis y1Ctriu F.Fl-egh(L y uui,calpain-cleaved subunit, RyR (FL) Full-length (F). C-terminus RyR1 against 2+ concentrations. 6 2 s.e.m. O 2 sidctd ape eeaaye ySSPG 4 es n immunoblotting and gels) (4% SDS-PAGE by analyzed were Samples indicated. as , api laaeo i ada R(–)adrbi kltlmsl SR muscle skeletal rabbit and (A–C) SR cardiac pig of cleavage Calpain naeae S-TLincreased GST-BT4L average, On oto) u h usqetadto f1 MGST-BT4L nM 10 of ( probability addition open increased subsequent substantially a in the resulted but control), iia fet( had alone cis GST effect the nM 100 to minimal where added channel a a sequentially shows 7B were Fig. GST-BT4L where chamber. experiments and out carried alone we moiety, GST GST the for control To odices in increase fold ( xeiet niae ermrfrainb h N-terminus the crosslinking by chemical formation whereas tetramer 2), indicated (Fig. using assays experiments tags co-IP by fusion self-interaction cells This mammalian disparate 1). HEK293 (Fig. in verified itself was with 1– strongly (residues BT4L interacts a region, 906), N-terminus coding of the RyR2 screening that entire revealed the Y2H sequence spanning its Our fragments that elucidate overlapping role. domain(s) of further functional RyR2 series to and the N-terminus structural identify the to precise with was interact study our potentially of aim The Discussion niaeta S-TLseiial nue statistically a induced specifically ( GST-BT4L significant that indicate ( alone GST n 1093 5 0canl) ycnrs,GTaoersle na1.7 a in resulted alone GST contrast, By channels). 10 gis y2Ctriu C,Ab (C), C-terminus RyR2 against P n y2Ntriu ermrzto 5047 tetramerization N-terminus RyR2 , 5 .5 nraei hne P channel in increase 0.05) hnes.Tetodtst oldtogether pooled datasets two The channels). 5 P o ( P n 5 o 5 channels). 7 .1 o S versus GST for 0.019 2142 P o y7.4 by gis y1N-terminus RyR1 against , 5 D -emnland N-terminal kDa 150 6 o . odrltv to relative fold 2.8 y8.6 by P o 5 P 6 .1 for 0.014 o 5 . fold 2.5 0.084). 6 0.4- Journal of Cell Science 0 Mfe Ca free nM 100 diint h aecanlo 0 MGTaoe olwdb 0n S-TL ntecscabr laent h ifrnei cl asbtenAadB. and A between bars scale in difference the note Please chamber. cis the in GST-BT4L, nM 10 by followed alone, GST nM 100 of channel same the to addition omattae urudn h eta orodai.Asmlfe oe fpooe y2Ntriu iscainidcdb xgnu TLpeptid BT4L exogenous by induced dissociation N-terminus RyR2 right. proposed the of on model shown simplified is A channel axis. ‘leaky’ fourfold a central in the resulting tetramerization. surrounding N-terminus tetramer RyR2 a of form model Proposed 8. Fig. by stabilized The further protein. native the be in residues cysteine between could bonds covalent tetramerization N-terminal and interactions, that into protein–protein suggest assemble findings non-covalent intrinsically These through can 4). domain tetramers Fig. N-terminus 3B; RyR2 (Fig. the DTT that tetramers the of form presence because to the able oligomerization, in still N-terminal was domain for N-terminus RyR2 essential discrete be appear to bonds disulfide not However, heart 5). N- (Fig. pig purified bonds involving tetramers disulfide native stable intrinsic the form the to of found in was N-terminus filtration Interestingly, the RyR2, tetrameric gel 4). (Fig. a by fragment observation, identified terminal this also with was Consistent species 3). (Fig. domain probability. open channel RyR2 enhances BT4L 7. Fig. 5048 ora fCl cec 2 (21) 126 Science Cell of Journal 2+ A hne ciiybfr n fe diino 0n S-TLt h i hme.()Canlatvt eoeadatrsequential after and before activity Channel (B) chamber. cis the to GST-BT4L nM 10 of addition after and before activity Channel (A) . igecanlrcrig fRR sn K using RyR2 of recordings Single-channel rwn ntelf sashmtcrpeetto fteRR -emnsitrcin that interactions N-terminus RyR2 the of representation schematic a is left the on Drawing miia vdnedrvdfo h urn iceia analysis biochemical current the from derived evidence empirical dimers. to lead only of site would single interaction a whereas monomer, per domains interacting two least tetramer because oligomer is any This (or expected. assembly be dimers would interaction of tetramers formation the than 2000), inter-oligomeric rather Lai, and Yin 2005; al., in et membrane (Yin two-dimensional lattice the involved within are If channels 8). channel adjacent were (Fig. between RyR2 fashion circular N-termini single closed a a the in within N-terminal other subunits each the with four associated where the model of a regions favors tetramerization observed u oe o y2Ntriu ermrzto ae on based tetramerization N-terminus RyR2 for model Our + stecag are at carrier charge the as . )sol nter emdae yat by mediated be theory in should 2) 2 0m ntepeec fcis of presence the in mV 60 e Journal of Cell Science uprstercnl rpsdNtriu oainsurrounding location N-terminus proposed and recently with, our the consistent contrast, entirely al., By supports 2007). is et al., model Liu et and/or tetramerization Wang 2002; N-terminal 2008; fusions al., al., et GST-RyR et Serysheva (Baker or 2001; docking GFP-RyR and modeling suggested of initially homology studies RyR the cryo-EM of periphery by the location at ‘clamp’ N-terminus proposed the the for with incompatible appears 8), (Fig. iufd od r o osre nRR res they else be or could in cysteines RyR1 these glutathionylation, in involved Alternatively, (e.g. RyR1. conserved modified in cysteines bonds. nitrosylation) post-translationally not disulfide N-terminal be are might for RyR2 bonds requirement disulfide the the but against Potentially, interactions, inter-subunit argues a N-terminal exclude with simply not RyR1 does agreement of this in presence However, 1997). the 5D,E) al., (Fig. et (Wu RyR1 report native previous the N-terminal for tetramers determined RyR. the empirically of 2001; centre the al., the are at with architecture et location RyR the Liu compatible of corners 2002; N-terminus the not the at placing al., region 2007) clamp appears al., et the et it within Wang (Baker Thus, 2008; al., 2010). studies et al., Serysheva initial et RyR1 (Tung the region the electron-dense that very clamp at the the to model by of due latter raised artifact inositol nature an the previously been of have of as might docking However, clamps structures colleagues, similar. quite and crystal (Serysheva is Tung domains the 2008) suppressor the al., empirically on and 2010), et structure core based al., binding the et was trisphosphate with (Tung that consistent crystallography model not X-ray is by (Baker the determined 2002) domain that al., oxidoreductase-like prediction 2008). an the et were al., forms on full-length et based N-terminus which the Serysheva generated RyR1 2002; model of N-terminus, al., the region et Although RyR1 clamp (Baker architecture the the RyR1 into docked for subsequently models (Tung colleagues structural of and sphere a Tung span by 2010). could highlighted al., protein, the et already GFP with long as or together insertion, of GST radius, the a use of of the side size either Furthermore, on native RyR. linkers the in flexible recombinant GST- occur not wild-type in do that changes or proteins solubilized fusion conformational GFP-RyR of and cause RyR freezing potentially might involving attributed GFP therefore RyR processing Additionally, or are N-terminus. sample the and GST of cryo-EM location centre) actual N-terminal RyR the as the from incorrectly effects. at result (e.g. allosteric the might insertions at long-range structure (e.g. periphery) changes induce RyR RyR conformational also RyR intrinsic distally-altered might the Hence, and perturbed of However have proteins locally 2007). discrete al., could both et GFP, or potentially Wang GST 2001; of GST al., insertion the et the of (Liu location insertion precise the GFP for or or evidence RyR2 as in wild-type taken to were differences at relative RyR3, maps or observed density RyR3 and cryo-EM of resultant respectively, N-terminus the extreme RyR2, the within at studies, tertiary previous fused the Ser437 the was at In its GFP 2010). other or al., of et GST each (Tung molecule docking to the four of adjacent precise the center placed immediately map a domains density EM crystal, N-terminal RyR1 native the the within remained Although in structure 1–559) 2010). acids monomer al., (amino et RyR1 a (Tung of RyR1 fragment N-terminal of the axis fourfold central the nieRR,w i o eetdslielne N-terminal disulfide-linked detect not did we RyR2, Unlike generate to used were methods computational studies, other In , 7kDa, 27 , 8nm omitrsbntdsliebnsmgtocri nioom or isoform- an in occur to might oxidation bonds cysteine disulfide might inter-subunit self-association, RyR1 form N-terminus and of RyR2 capable both be although all in However, determinants conserved isoforms. be the three therefore RyR3), could and tetramerization RyR2 N-terminus RyR1, of of 1–900 sequence ( residues N-terminus RyRs for of mammalian degree three residues the high cysteine(s) because between the homology Given buried possibly 1997). al., previously RyR1 et (Wu exposes purified folding solubilized, oligomers in altered in N-terminal only induced disulfide-linked on accessible were site Notably, becoming interaction states. sensitive, its disease conformation that normal suggesting is 2010; restore 2010), al., RyR2 can et al., Kobayashi and et 2009; al., RyR2 Uchinoumi et (R2474S) (Kobayashi function mutant inhibiting cardiac of or Paul- capable 2002; failing is al., dantrolene the et However, (Paul-Pletzer 2005). drug al., RyR2 et and this wild-type Pletzer bind 601–620) the to appear RyR1, residues not to RyR2 does binding to dantrolene of identical of observation is (RyR1 effects conserved 590–609 100% differential being has residues the site N-termini binding explain its RyR Despite to dantrolene. cardiac proposed and been differences skeletal previously conformational between disparate folding the local Interestingly, local disulfides. because into oxidation their RyR1 prevent in occluded xrse ihteNtriu rget TL ipae a co- displayed RyR2 BT4L, that fragment, observed We N-terminus role. , the structural with a expressed self- just than N-terminal the more mediating dissecting binding. in accurately domain central in involved versus interaction help sequences other truncated possibly with N-terminus specific small could of interactions series a weaker constructs of use significantly The domains. protein of RyR2 from alternative detracts transcription detection self-interaction An non-native BT4L gene the assay. robust the the interaction reporter that altered, protein to is the explanation Y2H N-terminus the prevents an the for or necessary of have sequence, access DPc10 precludes study in is that used It we conformation present constructs. that fragments domain domain central N-terminal central the notably RyR2 RyR2 AD4L screen the the the that Y2H of possible between any our interaction and However, any fragment detect 2009). and to al., 2442–2477 failed et acids Yamamoto, Yano and well- (Ikemoto amino 2002; 2460–2495) with acids RyR1 amino RyR2 previously (DP4; DPc10; short peptides central-domain-derived, suggested of synthetic been effects functional has documented domains central manner. tissue-dependent niaeta TLehne ciiya o Ca low at activity enhances BT4L that by indicate probability open channel al 1.A 0 MCa nM 100 At S1). Table ( concentrations splmnaymtra i.4.Gvntewd ra of array wide the it self-association, Given N-terminus with 4). was consistent N-terminus data BT4L Fig. its biochemical with exogenous particular material N- in Further, and (supplementary RyR2 oligomeric RyR2. with interact native the to found the of in associations terminus N- disrupting inter-domain subunit thereby partner, RyR2 binding endogenous local corresponding any functional its the for its requiring sequence with exerting not terminal channels, competing be direct, RyR2 might by is BT4L effects RyR2 purified proteins. on cytosolic recombinant effect other its and that native suggesting both with wfl nraei [ in increase twofold ti paetta y2Ntriu ermrzto a have may tetramerization N-terminus RyR2 that apparent is It and N-terminal RyR the between association intimate An y2Ntriu ermrzto 5049 tetramerization N-terminus RyR2 # 5 M Fg n upeetr material supplementary and 6 (Fig. nM) 250 3 ]yndn idn tlwCa low at binding H]ryanodine 2+ xgnu TLicesdthe increased , BT4L exogenous , ihfl Fg ) hs results These 7). (Fig. eightfold 2+ , concentrations, 5 similarity 85% 2+ Journal of Cell Science elnce n ls ed eermvdb etiuaina 1500 at centrifugation by removed were beads 4 glass at (425–600 minutes beads and 10 glass of volume nuclei half a Cell through suspension cell the dispersing htteNtria eini novdi Ca in involved is region prospect N-terminal suggested this study recent and the a plausible Indeed, that very investigated. further is be CPVT findings, should present of in the tetramerization pathogenesis of N-terminus the light RyR2 of In involvement 2011). potential al., the et Thireau and 2009; (Blayney Lai, arrhythmias fatal and after-depolarizations delayed abtseea uceS (10 SR muscle skeletal RyR1/2 Rabbit native of cleavage Calpain 0.6 G, (23 7.4 needle pH HEPES, a mM through 5 passages sucrose, 20 M by 0.3 inhibitors) (in protease ice and on homogenized were cells HEK293 assays crosslinking Chemical [ (Merck), Calbiochem from calpain-2 and high from CHAPS (Complete) Technologies), cocktail other inhibitor Roche, (Life all protease Technologies), Bio-Rad, Invitrogen from (Life reagents Invitrogen from electrophoresis from (HiMark) obtained markers were weight molecular reagents culture Cell Materials Methods and Materials low at closed channel RyR2 the maintaining Ca in role enhancing functional in BT4L exogenous N- interpretation of activity. likely action channel native most of RyR2 the rather mechanism the is that the this thus of note its for evidence, We direct N-termini, destabilization from 8). being (Fig. than three or subunit interactions other inter-subunit disruption this terminal the BT4L in of so, with resulting doing domain In association subunits. N-terminal tetrameric four with the the is of interaction its displaces one of of site N-terminus primary the interacts the BT4L RyR2, exogenous full-length when with that predict to reasonable seems nacscanlatvt tdatlcCa interactions diastolic N-terminal at inter-subunit activity channel of enhances disruption inter-subunit region. N-terminal RyR2-specific, the four Third, within for specifically the tetramers. located promoting evidence of bonds stable disulfide in present into N-termini assemble role we the channel that Second, direct a within find a subunits we RyR2 plays First, that closure. channel locus RyR2-associated structural in RyR2 defective 2012). be al., et could (Tang cardiomyopathies which termination, 5050 lgmrcitrcin ihnteRR -emnscudla to lead Ca could N-terminus SR RyR2 the within interactions oligomeric eetv -emnlttaeiainhsa motn oein role important that an suggest has pathophysiology. therefore RyR2 tetramerization We N-terminal phenotype. ‘leaky’ defective a with consistent y2baigCV uain r nw orsl n‘hyper- Ca in result diastolic to and known channels are mutations sensitive’ CPVT bearing RyR2 .1 rmpeo le H68 n ape eete nlzdby analyzed then were samples and EDTA, mM 6.8) 5 pH SDS- glycerol, 10% Ab and Blue, SDS, using with hydrazine immunoblotting 2% incubated 2% Bromophenol Tris-HCl, then mM and with (60 0.01% minutes stopped buffer 30 was loading for PAGE DTT Reaction mM glutaraldehyde. 10 without 0.0025% or with incubated eo egn 1 MDTo MH mM 1 or DTT mM (10 reagent redox eknEmr ytei 1-palmitoyl-2-oleoyl- synthetic Perkin-Elmer, 2 MKl H74.S eilswr eoee t20,000 at recovered were vesicles SR 7.4). pH KCl, mM 120 ih4uiso api- o iue tro eprtr.Poelsswas Proteolysis temperature. using immunoblotting room by at analyzed minutes and 2 buffer for loading calpain-2 SDS-PAGE with of stopped units 4 with y antibodies. RyR rmAat oa iis l te egnsfo Sigma. from reagents other all Lipids; Polar Avanti from 4 ˚ ,rsseddi rs ufrsplmne ih2m CaCl mM 2 with supplemented buffer fresh in resuspended C, hs tbeNtriu lgmrzto slkl opa a play to likely is oligomerization N-terminus stable Thus, nsmay ehv dniidteNtriu sa important an as N-terminus the identified have we summary, In 2+ codnl,cniin htwae rdsutthese disrupt or weaken that conditions Accordingly, . 2+ ora fCl cec 2 (21) 126 Science Cell of Journal ekdrn h isoi hs ncricmyocytes. cardiac in phase diastolic the during leak ˚ ,adtespraatwsrtie.Cl ooeae(20 homogenate Cell retained. was supernatant the and C, Myc . m )o i ada R(100 SR cardiac pig or g) 2 O 2 o 0mnts(n1 MNa mM 10 (in minutes 30 for ) sn 2+ -glycero-3-phosphoethanolamine ek fe edn to leading often leak, 2+ m )wstetdwith treated was g) 3 g ]yndn from H]ryanodine concentrations o 0mntsat minutes 10 for 2 6 n incubated and 2+ 5m)and mm) 25 release 2 m PIPES, )was g) g m m). for ufr a eaae ne dnia odtos leuinfatoswere fractions elution ml 2 Ab using conditions, immunoblotting identical by analyzed under and collected separated was buffer, otie ucinlRR channels. RyR2 functional contained n MET,HDAadNA(acltduigMxhltrsoftware, MaxChelator using (calculated NTA and HEDTA EGTA, www.stanford.edu/ mM 1 [ and nM 8 using performed was binding Ryanodine [ NaH mM (50 buffer against Pierce) cut-off, 12 at hours 18 ( bacteria for in expression Protein GST-BT4L 6 Healthcare). of C-terminal filtration with gel and GST-BT4L purification, and expression Protein rie gis eius14–35 a endsrbdpeiul (Jeyakumar previously described Ab consensus been RyR has 2001); al., 1344–1365) et 2005; residues against Lai, (raised and (Zissimopoulos SR previously transfection, Ab RyR1-specific described and 2006). as culture al., et out cell Zissimopoulos immunoblotting carried HEK293 and assays were system, pull-down GST co-immunoprecipitation, two-hybrid preparation, yeast The methods Other symmetrical re-establish to perfused was of chamber aliquots cis of K the addition the mM fusion, the to 210 by After added was induced KCl. RyR2 was M Purified incorporation 3 7.2. channel pH and KCl, chamber mM cis 210 HEPES, mM 20 containing across formed were 200- phosphatidylethanolamine a using bilayers phospholipid Planar recordings Single-channel were 4 at plasmid hour expression 1 at cDNA for pelleted rotor) RyR2 50.2Ti microsomes human and with homogenized transfected cells HEK293 RyR2 recombinant of Purification u ...9sieuigasml w tt cee(closed scheme state two the simple of a algorithm using modeling suite hidden-Markov 1.5.0.39 the were data using QuB www.qub.buffalo. recording idealized Buffalo, of (SUNY, were software seconds Currents displayed QuB of edu). 120 using and least effect channel at kHz the each RyR2, for 20 characterize on analyzed To at DTT) mM Corporation). digitized (+10 (Bruxton kHz, GST-BT4L software 5 5.0.1 at Acquire filtered with low-pass were fluctuations T,p .) reCa Free 7.4). pH DTT, eodn at recording h noul aeilwsrmvda 14,000 at removed was material insoluble the t4 at at spun was gradient ehrs G elhae o or t4 at hours 2 for Healthcare) (GE Sepharose 00 akile l,19;Zsioolse l,20) y2seii Ab RyR2-specific 2007); al., et Zissimopoulos 1997; al., et Mackrill 2000; diino MET,HDAadNAta hudcaecanlatvt;cis activity; channel cease should that NTA Ca and HEDTA free EGTA, mM 1 of addition he reeta n oiaincce,adteislbemtra a eoe at removed was material insoluble the and 10,000 cycles, sonication and freeze-thaw three .5m CaCl mM 0.15 sn S70sanr(i-a)adQatt n otae(Bio-Rad). software One Quantity and (Bio-Rad) scanner performed was GS-700 analysis Densitometry a Biotechnology. 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MET,2m T,p .,adpoes niios and inhibitors) protease and 7.4, pH DTT, mM 2 EGTA, mM 0.1 , 1093 ˚ , 2 ih01m isopropyl mM 0.1 with C IE,1MNC,06 HP,06 phosphatidylcholine, 0.6% CHAPS, 0.6% NaCl, M 1 PIPES, pto/onod.t) on [ Bound cpatton/downloads.htm). , a asdaantrsde 4447 Ftsmose al., et (Fitzsimmons 4454–4474 residues against raised was 2+ 100,000 2 PO ocnrto a ufrduigacmiaino CaCl of combination a using buffered was concentration b 4 ayae loo eyrgns,BAadcarbonic and BSA dehydrogenase, alcohol -amylase, 2149 H74 0m T n rtaeihbtr)by inhibitors) protease and DTT mM 10 7.4, pH , ˚ ˚ .Tespraatwsicbtdwt glutathione- with incubated was supernatant The C. .Teple a ouiie o ora 4 at hour 1 for solubilised was pellet The C. 6 g H300 i a a rae yPRi GXP (GE pGEX6P1 in PCR by created was tag His a asdaantrsde 9344 fRyR2, of 4933–4948 residues against raised was 2,0 p,Bcmn3T oo)fr1 hours 18 for rotor) 32Ti Beckman rpm, (28,000 2 . MET,2m T,p .) The 7.0). pH DTT, mM 2 EGTA, mM 0.3 , rie gis -emnl30rsde)was residues) 300 N-terminal against (raised ˚ i 5m IE,1MKl mM 2 KCl, M 1 PIPES, mM 25 (in C 2+ E.coli ˚ , .Ca n h atrdGTB4 was GST-BT4L captured the and C b 100,000 2 Dtiglcoiea OD at -D-thiogalactoside PO 2142 2+ g oet,Nvgn a induced was Novagen) Rosetta, 4 3 ¨ eednewstse ycis by tested was dependence 0 MNC,5mM 5 NaCl, mM 300 , o 0mntsa 4 at minutes 30 for ]yndn n 200 and H]ryanodine GST T PC(EHealthcare) (GE FPLC KTA a asdaantresidues against raised was 3 , ]yndn a separated was H]ryanodine g . 62%scoefraction sucrose 26–28% m 2,0 p,Beckman rpm, (28,000 ) uiidi h same the in purified g), / ? open). m unlabelled M 600 ˚ .The C. m 5 ˚ gof Cin 0.8. ˚ M D2 C. b 2 - r Journal of Cell Science a,F . ir,M,X,L,Sih .A n esnr G. Meissner, and A. H. Smith, L., Xu, M., Misra, A., F. Lai, T. Yamamoto, and N. Ikemoto, ht .B,Za,J,Zn,W,Ble .W,Tksia . ir .G n Ma, and G. W. Wier, H., Takeshima, W., C. Balke, W., Zang, J., Zhao, B., M. Bhat, oaah,S,Yn,M,Uhnui . utm,T,Ss,T,Oo . u X., Xu, M., Ono, T., Susa, T., Suetomi, H., Uchinoumi, M., Yano, S., Kobayashi, X., Xu, M., Mochizuki, H., Tateishi, M., Ono, T., Suetomi, M., Yano, S., Olivey, Kobayashi, P., Chang, O., J. McIntyre, S., D. Cheng, L., Ballester, H., L. Jeyakumar, ht .B,Za,J,Tksia .adM,J. Ma, and H. Takeshima, J., Zhao, B., M. Bhat, xrse sma values mean as expressed ae,M . eyhv,I . ecr . u . ute .J,Jag . Hamilton, W., Jiang, J., S. Ludtke, Y., Wu, S., Sencer, I., I. Serysheva, L., M. Baker, ere .H,Jni . hms .L,Sot,M,Wles . ilas .J. A. Williams, N., Walters, M., Scoote, L., N. Thomas, H., Jundi, H., C. George, S. Fleischer, hn .R,Ary .A n aLna,D H. D. MacLennan, and A. J. Airey, R., S. Chen, A. F. Lai, and M. L. Blayney, h xeietldt;LHC n ..cnrbtdraet and reagents contributed S.F. analyzed and and performed C.V., L.H.C. N.L.T. S.Z., data; S.M., design; experimental R.S., the study I.C., wrote the to J.W., and contributed B.C., experiments A.J.W. M.S., the and designed F.A.L. study, paper; the conceived S.Z. contributions Author Student’s unpaired using performed was analysis Statistical rcn-ak,P,Goaeea .A,Gla,C . ike,R . Hidalgo, T., R. Dirksen, P., C. Gilman, A., S. Goonasekera, P., Aracena-Parks, References Research at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.133538/-/DC1 Heart online available Wales material Supplementary I.C. and and J.W. S.Z.]; to Scholarships to Fellowship Placement Foundation Training FS/08/063 Heart Institute British number a by [grant supported was work This Funding manuscript. the edited F.A.L. and S.Z. materials; ere .H n a,F A. F. Lai, and H. C. George, and A. F. Lai, E., Rodriguez, A., J. McRoberts, I., Gukovsky, J., T. Fitzsimmons, receptor-Ca J. o ainn yetema niisctcoaiegcplmrhcventricular polymorphic model. mouse catecholaminergic knock-in al. 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