ise nldn ri n er Jnshe l,20) KCNQ1 2004). al., et (Jentsch heart different and in brain functions including important tissues play that members five comprises 96 ebneadKs,20;Snuntie l,1996). syndrome al., QT to long et lead causing genes Sanguinetti arrhythmias KCNE1-encoding al., cardiac or 2005; life-threatening et KCNQ1- Kass, either (Barhanin in and potential Mutations action Nerbonne cardiac 1996; the of repolarization nvriyo ea elhSineCne,Hutn X700 USA. 77030, TX Houston, Center, Science Health Texas of University Ato o orsodne([email protected]) correspondence for *Author Israel. ogQ uain tteitraebtenKN1hlxCand C Dvir Meidan helix KCNQ1 between interface disrupt the KCNE1 at mutations QT Long ARTICLE RESEARCH ß eevd2 oebr21;Acpe uy2014 July 8 Accepted 2013; November 25 Received 5 4 1 K voltage-gated of subfamily (Kv7) KCNQ The INTRODUCTION KCNE, KCNQ, QT, Arrhythmia, Long channel, Potassium WORDS: KEY the generates co-assembly KCNE1 and KCNQ1 ABSTRACT vv698 Israel. 69978, Aviv nKN1hlxCldt erae urn est n a and density current decreased a (PIP impaired from to arising phosphatidylinositol-4,5-bisphosphate mainly led activation, channel of C shift depolarizing helix disrupted mutants KCNQ1 mutations LQT interaction. in LQT intersubunit KCNQ1–KCNE1 All internal this the at interface. located intersubunit mutations LQT intracellular of mechanisms molecular unknown CE itlCtriu,teLTmtto 17 suppressed P127T the mutation of upregulation LQT cAMP-mediated the yotiao-dependent C-terminus, distal KCNE1 ihsgaigpoen oaheerglto upon regulation achieve to proteins signaling with hs CQ ei sipratfrcanlmdlto yPIP by S27. modulation channel at for phosphorylation important is KCNQ1 C reduced helix KCNQ1 by Thus, caused was which yotiao protein, the anchor brings repolarization. A-kinase AKAP9), as The potential syndrome known QT fibrillation. (also action long atrial cause cardiac and genes (LQT) the corresponding their to in Mutations crucial is which uuiscnitrc ihayoeo h ieKN auxiliary KCNE five the of one any with interact can subunits hra h CE itlCtriu per seta o the for essential appears C-terminus of distal regulation KCNE1 the whereas ihteKN1dsa -emns epsuae htti interface this for that crucial postulated We is C-terminus. interacts distal C helix KCNE1 KCNQ1 the that with shown have we Recently, stimulation. rble l,21) oasml fKN1wt KCNE1 with KCNQ1 2012; of al., et Co-assembly the Sun 2012). 2011; generates al., Kubo, and et Nakajo Wrobel 2001; characteristics al., functional et channel (Abbott different in resulting subunits, lfPongs Olaf eateto hraooy oubaUiest,NwYr,N 02,USA. 10027, NY York, New fu University, Institut Columbia Pharmacology, of Department 69978, Aviv, Tel University, Aviv Tel Pharmacology, & Physiology of Department 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,34–95doi:10.1242/jcs.147033 3943–3955 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 2 eateto iceity&MlclrBooy e vvUiest,Tel University, Aviv Tel Biology, Molecular & Biochemistry of Department ¨ hsooi,Universita Physiologie, r I KS I I KS 5 KS 1 oetKass Robert , 3 o Strulovich Roi , I eateto nertv ilg n Pharmacology, and Biology Integrative of Department yyta-eitdPAphosphorylation. PKA yotiao-mediated by KS hne ouain ee eeaie h yet the examined we Here, modulation. channel K + urn hti ia o h proper the for vital is that current ¨ e arads 62 obr,Germany. Homburg, 66424 Saarlandes, des t 4 I olA Hirsch A. Joel , KS 2 I aaSachyani Dana , KS hne ope together complex channel euainb K n PIP and PKA by regulation 2 ouain nthe In modulation. ) + I KS hnes(Kv) channels b 2 1-adrenergic n enr Attali Bernard and I K KS + 2 na e-a Cohen Ben-Tal Inbal , current, current, a b 2 - - , tde aesonta ei om ermrcparallel tetrameric phosphatidylinositol-4,5-bisphosphate a 2008). (PIP by modulation al., forms et Wiener D 2007; helix al., et that (Howard crystallographic coiled-coil shown and distal tetramerize, the proximal have D whereas helix comprises (CaM), studies and calmodulin C C-terminus bind helix that KCNQ coiled-coil B and The A Wiener 2006; 2008). helices al., (Ghosh et al., Shamgar trafficking 2008; Attali, et and and Haitin assembly 2006; KCNQ1 al., gating, be et The to channel shown 2008). been for al., has which important et C-terminus, large (Peroz a possesses fibrillation subunit atrial and (LQT) emn- n emn-–emn- nrclua ikr and linkers PIP in involved intracellular being as segment-2– segment-4–segment-2 C-terminus the proximal at and KCNQ1 in segment-3 residues basic of clusters identified ta. 01 ada ta. 03.KN1wsfudt increase to found was KCNE1 2013). al., PIP et Zaydman 2011; al., et D43adaeyaeccaeA9(lokona ADCY9) phosphodiesterase as known PP1, following (also regulation AC9 phosphatase achieve cyclase to adenylate protein and PDE4D3 PKA, kinase 08;hwvr eetwr niae htti Bhlxlne is al., linker helix et PIP AB (Hernandez for this required B not that indicates and work recent A be a helix however, to connecting 2008); suggested linker the been in has located channels KCNQ2–KCNQ4 on binding -emnspoie taei ltomfor KCNE1 the platform and strategic C a helix KCNQ1 provides hypothesized between We C-terminus 2009). interface al., the et (Haitin interaction that subunits interface this new two for a the important revealing coiled- between thereby is the C-terminus, interaction KCNQ1 that distal KCNE1 of shown direct with module previously a C have helix We 2009; found coil al., 2010). et have al., (Haitin et KCNE others and KCNQ1 Zheng KCNQ1 is and of Lundby KCNQ1 C-termini We of 2007; the of between al., 2013). et N-terminus N-terminus al., (Lopes the phosphorylated et at the be to S92 predicted 2004). at also al., S27 et (Kurokawa of dephosphorylation xie ace smrel eue yctslcapiainof application cytosolic by reduced PIP markedly is patches excised cst tblz h hne pnsae(apradSaio 2007). Shapiro, and (Gamper state open channel the stabilize to acts codnl,tesotnosrnonof rundown spontaneous the Accordingly, utmmrnu iei h CE -emnsi e structural PIP key a of determinant is C-terminus KCNE1 the in site juxtamembranous PIP neatn ihsgaigpoen Hii n tai 2008). Attali, the or yotiao and brings called (AKAP) AKAP9 (Haitin protein anchor proteins A-kinase scaffolding The signaling with interacting ytepyia neato fyta ihtedsa ei ,which Marx D, helix 2012; distal mediated the al., is with yotiao regulates modulation et of The interaction Li 2009). physical al., 2004; the by et al., Terrenoire 2002; et al., Kurokawa et 2005; Kass, and h CQCtriu loapast eacuilrgo for region crucial a be to appears also C-terminus KCNQ The h CQ -emnsas rvdsa nefc for interface an provides also C-terminus KCNQ1 The 2 2 2 bnigst lormiseuiefrKN1 eetstudies Recent KCNQ1. for elusive remains also site -binding Lusur ta. 03 ake l,20) h ieo PIP of site The 2005). al., et Park 2003; al., et (Loussouarn estvt 0-odoe h KCNQ1 the over 100-fold sensitivity 1, * I KS 1 oiHaitin Yoni , hne ciiyfloigpopoyainor phosphorylation following activity channel 2 2 estvt L ta. 2011). al., et (Li sensitivity euaino CQ Avre l,21) The 2012). al., et (Aivar KCNQ2 of regulation I KS hne ope oehrwt protein with together complex channel 2 1 amnDessauer Carmen , b arnri tmlto (Chen stimulation -adrenergic I KS a sbntaoeada and alone -subunit hnesosre in observed channels 2 idn (Thomas binding I KS 3 , 2 ,which ), channel 3943 a 2

Journal of Cell Science htteitaellritraebetween proper interface reveal for intracellular data crucial the the together, Taken that S27. at phosphorylation KCNQ1 Q uain oae nti nrclua nefc ewe the between interface of intracellular mechanisms this I unknown in located yet mutations the LQT explored we Here, modulation. ARTICLE RESEARCH 3944 LQT To 1A). underlying LQT5 (Fig. KCNE1 the of examined C-terminus mechanisms distal first the we at C-terminus, the KCNQ1 located mutations KCNE1 between the interface explore and C intracellular helix the to in located quest mutations our In I RESULTS neato ihKN1hlxCadsprsino cAMP- of suppression and C weaker helix showed mediated KCNQ1 P127T mutation with LQT interaction the C-terminus, KCNE1 uat tteKN1C-terminus KCNE1 the at mutants est n otg ih-hf fcanlatvto,which PIP activation, reduced channel by current of for decreased accounted right-shift be voltage displayed could a They and interaction C-terminus. density weaker showed KCNE1 R562M with and R555C R555H, K557E, KS KS urnsrsligfo Q5mttosadtruncation and mutations LQT5 from resulting currents a and - I b KS sbnt.LT uat nKN1hlxC S546L, C, helix KCNQ1 in mutants LQT1 -subunits. urn peuainta scue yareduced a by caused is that upregulation current I KS hne function. channel 2 a idn.I h distal the In binding. and - b sbnt is -subunits CQ emn ,A4V(etnkwe l,20) The 2004). al., to WT similar et very of currents in those (Westenskow resulted mutation A341V LQT5 V109I KCNE1 6, in mutation second segment a with proband KCNQ1 certain a has in it associated but been 2000), also al., QTc et (Splawski causes syndrome borderline mutation Romano-Ward P127T 2003; severe distal a al., a more et The (Ackerman 2001). with phenotype al., mild et a Schulze-Bahr mutation LQT5 with family missense KCNE1 a in two heterozygous interval the a described of been has as mutant one V109I The either P127T. and V109I with mutations wild- KCNQ1 cells CHO (WT) in co-expressing of type effects the probed we end, this itlCtriu Hii ta. 09.Dlto fteKCNE1 the KCNE1 of Deletion the 2009). and al., C-terminus et KCNQ1 (Haitin C-terminus the for distal essential between is module contact C previously helix the have coiled-coil two KCNQ1 We the C-terminus. spanning that KCNE1 shown mutants the truncation of regions with different channel KCNQ1 of WT expressing dependence voltage S1). Table the material supplementary moderate 1B,D,G,H; of (Fig. a activation change produced substantial mutant no LQT5 P127T ( KCNE1 The S1). < et eeaie,frcmaio upss h fet fco- of effects the purposes, comparison for examined, we Next, 0) lhuhsgiiat euto ftecretdniywith density current the of reduction significant, although 40%), ora fCl cec 21)17 9335 doi:10.1242/jcs.147033 3943–3955 127, (2014) Science Cell of Journal i.1 fet fKN1Ctriu eein n LQT5 and deletions C-terminus on KCNE1 mutations of Effects 1. Fig. otg J eainhp fW CQ+TKN1o deletion or KCNE1 ( KCNQ1+WT WT mutants of relationships (J) voltage otg H eainhp fW CQ+TKN1o LQT5 or KCNE1 KCNQ1+WT ( WT mutants of relationships (H) voltage to s 1.5 for from for s mV 3 +100 for stepped was voltage deletion KCNE1 C-terminus C-terminus with distal or the mutant (D) at LQT5 P127T mutants (B), mutant KCNE1 LQT5 WT (C), with V109I C- co- cells the KCNQ1, CHO in WT in deletions of expressed the traces and current mutations Representative LQT5 terminus. the of location the I KS Fg BCGH upeetr aeilTable material supplementary 1B,C,G,H; (Fig. n n 5 5 D 2 7–10). 11) urn–otg I n conductance– and (I) Current–voltage 11–17). D 97 F.Clswr edat held were Cells (F). 69–77 0m.Cretvlae()adconductance– and (G) Current–voltage mV. 60 97)i 0m nrmnsadte repolarized then and increments mV 10 in 69–77) I KS currents. A ato fKN1 indicating KCNE1, of cartoon A (A) D 2 0–2 E rteproximal the or (E) 109–129 0m o+0m o to (or mV +60 to mV 50 2 0m.Membrane mV. 90

Journal of Cell Science mean t eakydtriato PIP of determinant key a be ( C-terminus proximal ya+91m ih-hf fteconductance–voltage ( the kinetics S1) of deactivation Table right-shift faster material supplementary mV and 1B,F,I,J; +39.1 (Fig. relationship a ( by ( C-terminus reduction drastic proximal a KCNE1 in contrast, the In the 1J). of of Fig. deletion right-shift mV; moderate (+15 a relationship produced conductance–voltage it However, 1B,E,I). (Fig. ( C-terminus distal ARTICLE RESEARCH eesr n ufcett bls h hsclinteraction physical the abolish to is sufficient C helix KCNQ1 and the of membrane deletion necessary plasma that shown the have to does we trafficking but Previously, channel C-terminus the KCNE1 affect the not and interaction C the helix disrupts KCNQ1 mutation between LQT5 P127T KCNE1 The on impact to deact 5 6 91 s.e.m., 6 sfrW and WT for ms 7 I KS n 5 hne aig(ie l,2011). al., et (Li gating channel 7–10, D 0–2)ddntafc h urn density current the affect not did 109–129) < D 97)icue ieta a on to found was that site a includes 69–77) 0)o h urn est accompanied density current the of 70%) P , 2 .0) hsdlto nteKCNE1 the in deletion This 0.001). estvt n steeoeexpected therefore is and sensitivity t D deact 97,respectively; 69–77, 5 525 D 97)resulted 69–77) 6 5m and ms 55 P Hii ta. 09,dlto fteKN1dsa C-terminus work distal KCNE1 previous the our of C- deletion with 2009), KCNQ1 ( agreement al., the et In to (Haitin 2E,G). binding (Fig. P127T unaltered terminus displayed WT, mutant of V109I C-terminus percentage KCNQ1 the a with markedly KCNE1 (as P127T, of We mutation, interaction 2B,C,E,G). LQT5 the (Fig. KCNE1 reduced severe C-terminus the WT KCNE1 that and the found His-tagged deletion of pulldown WT, purified mutants GST-tagged GST LQT5 purified subunits. using and two C-terminus performed the KCNQ1 of were interface experiments the protein at MBP–His mutations with not GST- ( and purified alone MBP– C-terminus that purified with KCNE1 assay interacts pulldown His-tagged specifically C GST helix Here, a KCNQ1 2009). by tagged al., et show (Haitin directly C-terminus coiled- KCNQ1 we the the of with C C-termini, interacts helix C-terminus coil KCNE1 KCNE1 and the that KCNQ1 suggesting recombinant purified between D , 0–2)ttlysprse h neato ihteKCNQ1 the with interaction the suppressed totally 109–129) 0.05; n ora fCl cec 21)17 9335 doi:10.1242/jcs.147033 3943–3955 127, (2014) Science Cell of Journal 5 n ,Fg A.Nx,w xmndteipc fteLQT5 the of impact the examined we Next, 2A). Fig. 4, 5 ;Fg EG,weesteml-hntp LQT5 mild-phenotype the whereas 2E,G), Fig. 3; hw nDadE epciey n omlzdto normalized and respectively, ( E, pulldown input and the D of in Quantification (F,G) shown row. lower shown are the Inputs in mutants. LQT5 or 67–129) (residues 352–622 (residues D His- C-terminus of KCNQ1 row) WT (upper tagged Representative pull-down (E) GST row. of lower immunoblot the in shown are (77–129). Inputs C-terminus KCNE1 GST-tagged by mutants C- 352–622 KCNQ1 (residues WT His-tagged terminus the of row) (upper pulldown ** r hw ntelwrrw C uniiaino the of ( Quantification input (C) to row. normalized lower pulldowns the in shown Inputs are diagram). in (E1CT mutants deletion C-terminus 352–622 His- (residues D the C-terminus of KCNQ1 row) WT Representative (upper tagged (B) pulldown row). GST (lower of input immunoblot the used, in were seen KCNE1 as His-MBP-tagged and His-MBP of ulon(S D uprrw fteHis-MBP- the of row) (upper PD) (GST pulldown C- KCNE1 and terminus. the C disrupt helix KCNQ1 mutations between LQT5 interaction and LQT1 2. Fig. -emnswt CQ ei ( C KCNE1 helix KCNQ1 of with in interaction C-terminus C, direct Helix the Q1 illustrating 535–572, diagram), (residues GST-tagged C by helix peptide, KCNQ1 His-MBP control or in E1CT diagram) 67–129, (residues C-terminus KCNE1 tagged 9–0)b S-agdW CE C-terminus KCNE1 WT GST-tagged by 396–504) 9–0,QC ndarm yGTtge KCNE1 GST-tagged by diagram) in Q1CT 396–504, P , .1 D ersnaieimnbo fGST of immunoblot Representative (D) 0.01. n 5 –) * 3–5). A ersnaieimnbo fGST of immunoblot Representative (A) P , .5 ** 0.05; 5 45% P , D 6 9–0)o LQT1 or 396–504) .1 *** 0.01; n 0 mean 10, n 5 5 ) * 5). ) qa amounts Equal 4). P P , , 0.05, 0.001. 6 s.e.m., 3945

Journal of Cell Science idn oteKN1Ctriu b 2.5 KCNE1 (by the C-terminus KCNQ1 of the ( deletion to binding contrast, C-terminus In proximal 2B,C). (Fig. C-terminus ARTICLE RESEARCH IFfursetsgaso F-agdW CQ n Q1mtns()ado F-agdW n Q5mtn F snraie ommrn inlarea signal membrane the to of normalized 3946 as Quantification (F) KCNQ1. mutant WT LQT5 with and WT co-expressed YFP-tagged co- mutant of cells and LQT5 expres CHO (E) or cells mutants of KCNE1 LQT1 CHO images WT and fluorescence of KCNQ1 YFP-tagged TIRF ( images WT (D) (C) CFP-tagged panels) KCNE1. of panels). (right with signals (lower fluorescent mutants fluorescence membrane. mutant+KCNE1 TIRF LQT1 TIRF plasma LQT1 C and G589D the helix CFP-tagged panels) to CFP-tagged or (left trafficking expressing panels) Epi-fluorescence channel (upper (B) the KCNQ1+KCNE1 GPI-citrine. affect WT and CFP-tagged not KCNQ1 do WT mutations CFP-tagged LQT5 expressing and LQT1 3. Fig. n con o h erae urn est bevdwt P127T. with observed density could current trafficking decreased channel the defective for P127T. whether account mutation examined LQT5 KCNE1 we severe First, the of function disrupted bandfo elepesn CQ–F Fg B upper 3B, intensity (Fig. epi-fluorescence KCNQ1–CFP expressing strong cell the a weak to from somewhat membrane relative obtained the comparing plasma signal by WT the TIRF illustrated of further retention to cytoplasmic is substantial KCNQ1 KCNQ1 The KCNQ1–CFP 2004). WT al., known et of that the (Kanki reflecting processing GPI-citrine, revealed of with partial slightly Co-transfection GPI-citrine only microscopy. colocalized and was TIRF intensity signal under KCNQ1–CFP GPI-citrine Nichols, the very and detected 3A, (Glebov express Fig. heavily cells in of to shown plasma membrane As known for 2004). plasma control the is at positive The TIRF GPI-citrine strongly a reflection controls. as expression. the used protein internal membrane negative was fluorescent validate (GPI-citrine) and total citrine positive (GPI)-linked To using used glycosylphosphatidylinositol microscopy. we imaged KCNE1– methodology, (TIRF) we and (KCNQ1–CFP fluorescently fluorescence which with subunits cells mutant YFP) CHO and transfected WT we tagged purpose, this For n 5 5 esuh oivsiaetemcaim neligthe underlying mechanisms the investigate to sought We ;Fg 2B,C). Fig. 5; 66) *** 16–60). P , .0.Saebr:10 bars: Scale 0.001. D 97)sgiiatyehne the enhanced significantly 69–77) m m. 6 0.2-fold, P , 0.001, peuaino the cAMP-mediated of the upregulation suppress C-terminus distal and KCNE1 mutation LQT5 P127T The ipae yteKN1P2TLT uatcudntbe not could density mutant current LQT5 in P127T decrease KCNE1 the the 3D,F), by (Fig. displayed above shown As hs nefc ntafcigcno ettlyexcluded. totally be cannot trafficking on effect an Thus, osntalwdsrmnto ewe h inl rmthe K from which some ER, signals for cortical relevant the the from especially arising between is those TIRF and discrimination However, membrane allow defects. plasma trafficking not by for does not accounted does be mutant to LQT5 to the seem by trafficking displayed density ( membrane current decreased channels mutant plasma WT LQT5 of comparable that KCNE1 showed YFP-tagged the P127T Importantly, microscopy TIRF 3B). under under (Fig. revealed obtained that signal to stronger relative the epi-fluorescence by confirmed further In is mutant 2004). to KCNQ1 compared P al., the signal (13% TIRF that et KCNQ1 weaker found much WT we a (Kanki exhibits work, been mutant in previous G589D (ER) retained previously this heavily with reticulum has is agreement a that endoplasmic as mutant channel used mistrafficked the this was a the C-terminus, as because in described KCNQ1 located control, the G589D, of negative mutation D helix LQT1 coiled-coil KCNQ1 The panels). , .0)(i.3,) h outE eeto fteG589D the of retention ER robust The 3B,E). (Fig. 0.001) ora fCl cec 21)17 9335 doi:10.1242/jcs.147033 3943–3955 127, (2014) Science Cell of Journal 6 A IFfursec mgso H el co- cells CHO of images fluorescence TIRF (A) fW CQ,mean KCNQ1, WT of 4 I KS n 5 current 66;Fg DF,sgetn htthe that suggesting 3D,F), Fig. 56–60; D + 0–2 eeina the at deletion 109–129 hnes(o ta. 2013). al., et (Fox channels 6 s.e.m., n 5 16–25, sing

Journal of Cell Science n PApopaaeihbtr de ntepthppte(Marx WT pipette 2002). patch al., the in et added inhibitor, phosphatase PP2A and ihW CE rismtns rnfce H el were cells together CHO yotiao, Transfected mutants. WT 200 its and by or stimulated KCNQ1 KCNE1 WT WT with both with transfected htotie ihteWT the to similar with was mutant obtained KCNE1 P127T that whether tested the we of Thus, modulation. cAMP-stimulation the disrupt might region this APpu kdi cdtetet(t+0m,70% mV, +50 (at treatment acid okadaic mean stimulation; plus cAMP h CE itlCtriu ol eipratfrcAMP- for that important hypothesized be the We could of C-terminus defects. upregulation dependent distal trafficking KCNE1 by the for accounted ARTICLE RESEARCH h idpeoyeLT 19 uatwssignificantly was mutant V109I LQT5 Interestingly, 4E,F). phenotype (Fig. mild stimulation acid the okadaic plus distal mutant plus KCNE1 cAMP the cAMP deletion Likewise, upon 4C,D). C-terminus stimulation (Fig. P127T treatment its acid the lost okadaic contrast, completely striking mutant In LQT5 observed. conductance– were and kinetics relations deactivation voltage in changes significant no 6 I KS m s.e.m., APad0.2 and cAMP M urnswr infcnl peuae by upregulated significantly were currents n 5 7, D I KS I 0–2 a nepnieto unresponsive was 109–129 KS P urn,adta uain in mutations that and current, , hne.COclswere cells CHO channel. .1 i.4,) however, 4A,B); Fig. 0.01; m kdi cd PP1 a acid, okadaic M 6 9 hncmae oW CE,teewsn significant no when was yotiao with there interact to KCNE1, KCNQ1 that of revealed ability WT the precipitate results in to the difference The in compared S1B). intensity when Fig. signal KCNQ1 material the the normalizing (supplementary by to yotiao, signal with KCNQ1 quantitatively yotiao of antibodies, interaction were anti-yotiao the mutants WT tested with antibodies. its probed both anti-KCNQ1 or blots with KCNE1 expressing Western immunoprecipitation WT cells plus to from yotiao, subjected WT Lysates and west S1A). KCNQ1 cells, to 293 Fig. HEK subjected material transfected in were proteins and lysates the C-terminus P127T of mutations expression distal the proper whether KCNE1 examined D first impaired in we the mutants, underlying modulation mechanisms possible cAMP-mediated the elucidate yotiao with To interaction KCNQ1 mutant the deletion disrupt and mutation LQT5 P127T peuae ycM lsoaacai ramn a 5 mV, +50 (at treatment acid okadaic plus 64% cAMP by upregulated 0–2 irpe CQ neato ihyta.T verify To yotiao. with interaction KCNQ1 disrupted 109–129 6 CE itlCtriu upestecAMP-mediated of the upregulation suppress C-terminus and distal mutation KCNE1 LQT5 P127T 4. Fig. eodnswt rwtotcM+kdi cdi h ac pipette, patch the in acid ( cAMP+okadaic respectively. without or with recordings to repolarization from s 3 200 of traces) (red presence cAMP+0.2 or traces) (black absence to repolarized then at and held were Cells (G). V109I APoaacai eesgiiat * by significant. induced were upregulation acid current cAMP+okadaic V109I KCNE1 the mV, +20 CQ+Tyta n TKN1()o CE 17 D or (D) P127T KCNE1 or (B) KCNE1 KCNE1 WT and yotiao KCNQ1+WT KCNE1 WT (C), and P127T yotiao KCNE1 KCNQ1+WT (A), WT KCNE1 co-expressing cells CHO from stimulation; 7 ora fCl cec 21)17 9335 doi:10.1242/jcs.147033 3943–3955 127, (2014) Science Cell of Journal D 2 0–2 F rKN1V0I() otg a tpe for stepped was Voltage (H). V109I KCNE1 or (F) 109–129 0m o+0m n1 Viceet olwdby followed increments mV 10 in mV +60 to mV 50 m kdi cd urn–otg eainhp fWT of relationships Current–voltage acid. okadaic M n 5 2 –) bv 3 V h WT the mV, +30 Above 6–7). I KS n 0m o . .Rdadbakcre represent curves black and Red s. 1.5 for mV 40 5 current. 6, 2 P 0m o . .Clswr eoddi the in recorded were Cells s. 1.5 for mV 40 , 2 .5 i.4G,H). Fig. 0.05; ersnaiecrettae recorded traces current Representative 0m n tpe o+0m o s 3 for mV +30 to stepped and mV 90 r ltig(supplementary blotting ern D 0–2 eeina the at deletion 109–129 P , D .5 ** 0.05, 0–2 E n KCNE1 and (E) 109–129 I KS D P 0–2 onot do 109–129 n,above and, , 0.01. m M 3947

Journal of Cell Science et estott xmn hte h CE itlC- distal KCNE1 the whether examine to out set we Next, cAMP- during S27 stimulation at dependent KCNQ1 of mutant deletion phosphorylation and impair mutation LQT5 P127T The C- S1B,C). distal KCNE1 mutant with deletion or terminus mutant LQT5 P127T with co-expressed ARTICLE RESEARCH 3948 min 10 for treated were mutants 250 yotiao. its or of WT with KCNE1 and presence WT and KCNQ1 WT the plus both yotiao, in expressing cells KCNQ1, 293 P127T HEK in Transfected S27 of mutations phosphorylation terminus .-odices nS7popoyain( phosphorylation S27 in significant increase a produced 2.6-fold cAMP-stimulation yotiao, with expressed CE rismtns nteWT the In WT mutants. either with its co-expressed or were when yotiao KCNE1 different WT significantly plus not KCNQ1 2003). under WT was al., S27 phosphorylation et at S27 (Kurokawa phosphorylation Basal conditions KCNQ1 cAMP-stimulated KCNQ1 of and of extent basal S27 the phosphorylated explore probed against to also antibody and specific were complex a blots anti-KCNE1 with the Importantly, in purposes. and expression quantification anti-yotiao protein for validating anti-KCNQ1, blots for and with antibodies blotting of western probed by analog were 0.2 followed SDS-PAGE permeable with to subjected membrane and a cAMP (8CPT), monophosphate i.5,) ncnrs,frteP2TLT uain the mutation, LQT5 P127T the in ( for increase contrast, treatment 2-fold In cAMP a 5A,B). mild- upon with Fig. the phosphorylation V109I, for found mutation S27 was LQT5 stimulation comparable phenotype A 5A,B). Fig. m -4clrpeyti)aeoie3 adenosine 8-(4-chlorophenylthio) M m kdi cd ellstswere lysates Cell acid. okadaic M D 0–2 splmnaymtra Fig. material (supplementary 109–129 I KS D hne ope co- complex channel 0–2 disrupted 109–129 n 5 n 5, 5 D 109–129 3, P 9 , ,5 P 9 0.0001; , -cyclic 0.05; n Tyta rdcdasgiiat24fl nraei S27 in increase 2.4-fold significant ( a KCNE1 produced WT phosphorylation both yotiao with that WT co-expressed show and K557E Results mutant KCNQ1 treatment. the cAMP following phosphorylation ei C helix n 52,lctdi h CQ ei Fg 6A; (Fig. C helix KCNQ1 the in located K557E R555H, R562M, R555C, S546L, mutations, CHO and in LQT1 KCNE1 five WT with co-expressing of cells effects the studied then We I 9,9%ad5%ihbto fcretdniya 6 V the mV; +60 at 88%, density current 75%, of S546L, inhibition respectively, D 57% (for R562M, activation, and 99% deactivation and channel 69%, K557E faster of R555H, and potent dependence R555C, kinetics by markedly voltage activation paralleled mutations the slower were which of All densities, right-shifts S1). current the Table reduced material supplementary neae Q1mtto K5E osnot. does an (K557E) whereas mutation regulation, LQT1 yotiao unrelated disrupts P127T mutation LQT5 the oe n o ttsial infcn 16-odincrease, (1.65-fold significant was statistically exposure not cAMP and following lower phosphorylation S27 in increase 2 hshrlto a bandwt h CE itlC- distal KCNE1 the mutant with deletion obtained terminus was phosphorylation S27 P eiigi CQ ei n hs eetivle weaker involves defect whose PIP and to K557E, mutant C mutation helix deletion binding LQT1 KCNQ1 the the in of whether residing examined that we than comparison, larger be D V109I to the appears of effect mutant the phosphorylation, acid-stimulated okadaic P KS . . V 0–2,atog o ttsial ifrn Fg C.For 5C). (Fig. different statistically not although 109–129, 50 .5.Smlry ekradnnsgiiatsiuainof stimulation non-significant and weaker a Similarly, 0.05). .5 i.5,) hnepesda h eaiecM-and cAMP- relative the as expressed When 5A,B). Fig. 0.05; urnsrsligfo Q1mttosa h KCNQ1 the at mutations LQT1 from resulting currents a 4 V 6. V 4. V 9. Vand mV +92.7 mV, +47.1 mV, +65.4 mV, +48 was ora fCl cec 21)17 9335 doi:10.1242/jcs.147033 3943–3955 127, (2014) Science Cell of Journal bec n rsneo 250 of presence and P127T) absence and ( (V109I truncated mutated WT, or co-expressing and lysates KCNQ1 cells WT 293 yotiao, HEK of (IB) immunoblots during S27 at stimulation. KCNQ1 cAMP-dependent mutant of deletion phosphorylation and impair mutant 129 P127T LQT5 5. Fig. ( uatK5Efo el rae nteasneand absence the 250 in of LQT1 treated presence or cells KCNQ1 from WT K557E co- and mutant lysates KCNE1 cells WT 293 yotiao HEK expressing of immunoblots and acid nue hshrlto fWT of of extent phosphorylation the induced where acid- phosphorylation cAMP+okadaic S27 relative stimulated the as S27 expressed KCNQ1 was phosphorylated of quantification alternative *** and (250 unstimulated cells both cAMP-stimulated for to blot) signal (anti-KCNQ1 phosphorylated input dividing KCNQ1 by KCNQ1 calculated row phosphorylated was (fourth of S27 KCNE1 Quantification and (B) top) top). from from from row row (third (second KCNQ1 yotiao top), top), from row (first phosphorylated S27 against KCNQ1 antibodies with probed were cd n omlzdt tmltdW KCNQ1+WT WT ( stimulated KCNE1+yotiao to normalized and acid) n 2 5 P n –) * 3–5). seblw,cudatrsiuaino S27 of stimulation alter could below), (see , 5 .0;n:ntsaitclysgiiat C An (C) significant. statistically not ns: 0.001; 4, P P , , D .5 s o ttsial significant. statistically not ns: 0.05; 0–2)KN1fo el rae nthe in treated cells from KCNE1 109–129) .1 i.5,) ecnld that conclude We 5A,B). Fig. 0.01; D n m 0–2 17fl increase, (1.7-fold 109–129 5 8CPT+0.2 M –) * 3–5). P , m 8CPT+0.2 M .5 ** 0.05, m m I 8CPT+0.2 M A Representative (A) KS kdi cd Blots acid. okadaic M a qa o1 to equal was P , 0.01, m okadaic M m okadaic M D n n 109– 5 5 5, 5,

Journal of Cell Science uat oepesdwt TKN1ehbtdsimilar LQT1 exhibited TIRF C KCNE1 with helix WT CFP-tagged trafficking with the channel that co-expressed on found mutants we impact microscopy, the Exploring R562M EERHARTICLE RESEARCH CE -emnswe oprdt TKN1C-terminus P KCNQ1 S546L WT WT: of to C (percentage compared the helix when with C-terminus interaction KCNQ1 KCNE1 weaker all significantly exhibit that mutations LQT1 revealed experiments to GST-pulldown trafficking channel membrane the affect plasma with not the interaction do the but disrupt C-terminus mutations KCNE1 LQT1 C helix The 720 was the above, as mutants respectively, mV, +40.7 1.36 n 194 and t respectively, deact , .5 R555H 0.05; 6 .7s 1.28 s, 0.07 a 675 was 5 6 6 52% 8m,respectively, ms, 18 0ms, 50 P 6 6 , 8, 5 6m,550 ms, 76 .5 o Tadtemtnsa bv,the above, as mutants the and WT for 0.05; n 45% 5 6 P ;alrslsaemean are results all 6; , .7s 1.40 s, 0.07 6 .1 mean 0.01; 3, n 5 t 1/2 5 P 41% 6 , 5–10, 6m,428 ms, 36 P .0;K557E 0.001; a 0.91 was , 6 .5 nmtn R562M, mutant in 0.05; 5, 6 6 P .1sad1.41 and s 0.11 P , s.e.m.; , .0;frW n the and WT for 0.001; .0;R555C 0.001; 6 6 .7s 1.20 s, 0.07 6 6m,460 ms, 26 5 ... Fg 6). (Fig. s.e.m.) n 25% 5 ;Fg 2D,F). Fig. 3; 6 8, 5 6 6 P 65% 6 , .9s, 0.09 .6s, 0.06 9ms 29 t 0.01; deact 6 8, rfikn oWT to trafficking edt eue urn est n oetrgtsitof right-shift the an potent involve that might KCNQ1–PIP mutations a hypothesized and C impaired helix and residue we these underlying density basic relations, mechanism a current conductance–voltage neutralize reduced the study to this lead in mutations LQT1 kmr ta. 09.Fg Asosasadr protocol standard a shows 7A Fig. 2009). 2008; al., al., et et (Hossain Okamura phosphatase voltage-sensitive Dr-VSP the Knie l,20) ecnld httedcesdcurrent decreased accounted the be cannot defects. that mutants trafficking LQT1 by conclude for five report We the previous by a 2004). displayed with density al., agreement in et is of (Kanki mutant membrane plasma LQT1 the R555C to trafficking the normal The 3C,E). Fig. 25; is rbdtePIP the probed first eius hc eepeiul ugse opa oein role a play to PIP suggested by basic previously of modulation cluster channel were a encompasses which C helix residues, KCNQ1 of half distal The impaired showed PIP C with helix interaction KCNQ1 in mutations LQT1 ora fCl cec 21)17 9335 doi:10.1242/jcs.147033 3943–3955 127, (2014) Science Cell of Journal CQ Q1mtnsc-xrse ihW KCNE1 WT with ( co-expressed mutants LQT1 and WT KCNQ1 of relationships conductance–voltage Current– and some shift. (H) depolarizing for voltage their applied of higher were because mV that mutations, +160 except LQT1 to 1, up Fig. of in voltages described membrane is that protocol as voltage-clamp same The R555H (G). the (D), R562M and R555C (F) WT (C), K557E with S546L (E), cells mutants CHO LQT1 in (B), co-expressed KCNQ1 KCNE1 current WT Representative of where (A). sequence, traces its situated and are C-terminus mutations the LQT1 in C on helix mutations of location LQT1 C helix KCNQ1 currents. of Effects 6. Fig. n 5 –)(I). 5–9) I 2 KS estvt fW n uatcanl using channels mutant and WT of sensitivity ato foeKN1sbntidctn the indicating subunit KCNQ1 one of cartoon A 2 2 hnest h lsammrn ( membrane plasma the to channels neato.T etti yohss we hypothesis, this test To interaction. 2 Pr ta. 05.A oto the of most As 2005). al., et (Park n 3949 5 I KS 8–

Journal of Cell Science ehd)a ewne opeetafs u-ono WT of run-down fast a prevent to wanted we as Methods) rc) eotie eaieylwrsprsino WT of suppression lower relatively a obtained current slight We a Dr-VSP trace). of absence 21% the of in overshoot while trace), red panel, EERHARTICLE RESEARCH 3950 our because al., Mg et contain Kruse not by did found solution that intracellular to compared currents WT monitor to used previously eunt 1 V(rs ta. 02.We hspooo was led Dr-VSP protocol of this activation min, 64% When a 2 a 2012). for a to by al., s then 10 et followed every and (Kruse applied phosphatase channel, repeatedly mV the +10 the activates to that opens return step prepulse voltage mV mV +100 +10 a where ( Dr-VSP of activation 6 euto fWT of reduction 4 6 a bevd( observed was 4 ai rerio Danio I I KS KS urns( currents urnsbfr n following and before currents S,as nw sTPTE), as known also VSP, n 5 ;Fg A etpnl red panel, left 7A, Fig. 5; 2+ n seMtrasand Materials (see 5 ;Fg A right 7A, Fig. 5; I I KS KS ol eepce orfettecanlafnt o PIP for affinity the channel activate the and reflect decline channel to current the expected of rate be open the would both sweeps, successive to pulse the order step During Dr-VSP. 2-s-long in a mV s) +100 5 to (every repeatedly applied we difficulty, otg eedneo ciain hc rvnste being them the prevents WT of like which mV right-shifts +10 activation, five potent at activated the of exhibit for dependence applied they be However, voltage because not 2007). could mutants Hille, protocol LQT1 and prepulse mV (Suh +10 channels this the with interaction opttvl euigteaon ffe PIP free of amount the reducing competitively lcrsai idn otengtv hre fPIP of charges by currents negative KCNQ the the Mg to reduce Indeed, Dr-VSP. to binding of found absence electrostatic been the in previously even current, ora fCl cec 21)17 9335 doi:10.1242/jcs.147033 3943–3955 127, (2014) Science Cell of Journal 55,K5EadR6M respectively. R562M, and K557E R555H, ( 0.55 P fWT of traces current ( Representative trace) (B,C,E,G,I,K) trace) (red (black sweeps first last the and at Dr-VSP the of in presence application protocol to sensitivity min. WT 2 shows for panel s protocol Left 10 This every s). applied (3 repeatedly mV was and +10 Dr-VSP to activate return to mV then s) +100 (2 a step by followed voltage channel, the open to from s, (3 to mV stepped +10 first is where potential protocol membrane tri-pulse a of by means recorded Dr-VSP phosphatase voltage sensitive the to WT sensitivity of (KCNQ1+KCNE1) traces current Representative (A) Dr-VSP. voltage- phosphatase the sensitive C to helix sensitivity KCNQ1 greater in exhibit mutations LQT1 7. Fig. urn rcsa ie0(lc rc)adat and trace) (black 0 time at traces are current Shown Dr-VSP. activate and from channels s, (2 mV +100 2 to stepped membrane is where potential protocol, pulse recorded one Dr-VSP by to sensitivity R562M and ie1 rdtae.Kntc fcurrent of WT Kinetics for trace). decline (red s 10 time rdtae.RgtpnlsosWT shows panel sweeps Right last and trace). trace) (red (black first Dr- the of at absence VSP the in application protocol vrta eodda ie0 t1 ,rto of 0.74 ratios 0.75 s, were 10 decline At s 0. current 10 time at time recorded at that measured over currents as of expressed ratio was a and drop state current steady the before reached an s) at (10 point quantified time was early mutants LQT1 five the S.Tecmaio ewe WT Dr- between of comparison presence The or VSP. absence the in shown n , 0m odn oeta)t ohoe the open both to potential) holding mV 90 5 .1,0.54 0.01), 7, I 6 6 KS .4( 0.04 .2( 0.02 P I KS , seFg ) ocruvn this circumvent To 6). Fig. (see 56,R5C 55,K557E R555H, R555C, S546L, , .1 o T 56,R555C, S546L, WT, for 0.01) n n 5 5 6 7, ) 0.32 7), I .6( 0.06 KS P 2 n uatcanl are channels mutant and , 0m odn potential) holding mV 90 .5 n 0.48 and 0.05) n I KS 6 5 .1( 0.01 4, 6 estvt to sensitivity 2 .5( 0.05 P n , vial for available 5 n 0.05), 5 5). n 3, 5 I 6 2 KS I 5), KS Thus, . 2+ 0.04 and I has KS 2 ,

Journal of Cell Science oehbtdsgiiatylwrbnigt PIP to binding lower significantly exhibited to ipae ytehlxCLT uat rs rmterweaker their from arose mutants PIP LQT1 to C binding we helix phosphatase the Next, Dr-VSP by the 7D,F,H,J,L). displayed to (Fig. sensitivity higher the protocol whether assessed this following overshoot P 0.74 uatcanl eeaaye nteasneo rsneo Dr- WT of between comparison presence The or 7D,F,H,J,L). absence the (Fig. VSP in analyzed were channels mutant xeiet fprfe i-agdW n Q1mtn KCNQ1 PIP mutant using LQT1 C-terminus and WT His-tagged purified of experiments xiie infcnl atrcretdciecmae oWT to s compared 10 decline the time current R562M S546L, I faster at and significantly mutant K557E and a R555H, for R555C, exhibited trace) mutants Except that black indicate trace). results 7B,C,E,G,I,K, red that shown (Fig. over also 7B,C,E,G,I,K, s are 0 (Fig. was 10 traces and time time current Representative at state at 0. measured steady time currents reached at of time recorded ratio drop early a current an as at before expressed quantified s) was (10 (+KCNE1) point mutants LQT1 five suigta h aeo PIP of rate the that assuming ARTICLE RESEARCH h atri h aeo urn erae h oe h channel the lower the decrease, current of PIP rate for the affinity is faster the TKN1Ctriu pretg fW idn:43% binding: WT of [percentage C-terminus KCNQ1 WT S,WT VSP, epciey(l eut r mean are results (all respectively P KS , , .5,0.55 0.05), .1 o T 56,R5C 55,K5EadR562M, and K557E R555H, R555C, S546L, WT, for 0.01) 6 a 0s aiso urn eln ee0.75 were decline current of ratios s, 10 [at .2( 0.02 I n KS 5 2 2 6 ots hsasmto,w efre pulldown performed we assumption, this test To . ) 0.32 7), h ieiso urn eln o WT for decline current of kinetics The . n uatcanl eddt hwcurrent show to tended channels mutant and .4( 0.04 2 cae grs ed.W on l mutants all found We beads. agarose -coated 6 n 5 .1( 0.01 7, 2 P n esnhssi ogl h same, the roughly is re-synthesis , 5 6 .5 n 0.48 and 0.05) 3, ...] nteasneo Dr- of absence the In s.e.m.)]. P , .1,0.54 0.01), 2 scmae othe to compared as 6 6 6 .5( 0.05 .4( 0.04 .6( 0.06 I KS 6 I n the and KS n 2for 12 n n 5 5 5 and 5), 4, 7, CQ ei ,ecp 56,hv nipie neato with interaction impaired an have PIP S546L, except C, helix KCNQ1 mean 14% and h n ad h Q1mtnsi CQ ei (R555C, C helix KCNQ1 PIP in reduced showed mutants R562M) LQT1 and K557E the R555H, hand, one the Hii ta. 09.Hr,w hwta Q uain located mutations disrupted LQT interface module this that C of show sides helix we both Here, coiled-coil on 2009). KCNQ1 al., et the cardiac (Haitin and crucial KCNE1 the the C-terminus between a forming interaction distal the subunits at to corresponding the and located channel of mutations interface molecular the LQT intracellular into insights underlying novel provide mechanisms we work, present the In DISCUSSION tblzsteoe tt ftecardiac the of state open the stabilizes hpr,20;ShadHle 05 hn ta. 03.PIP 2003). al., et Zhang 2005; Hille, and Suh 2007; Shapiro, togrgtsito hne ciain nteohrhn,the suppressed hand, C-terminus, of other distal upregulation cAMP-mediated KCNE1 the yotiao-dependent in On P127T activation. mutation channel LQT5 of right-shift strong ebaepopoii PIP phospholipid membrane 8C). (Fig. S27 at phosphorylation KCNQ1 reducing eaiesiti h otg eedneo activation of dependence voltage the in producing and shift kinetics negative deactivation a slowing thereby channel, o R555H, for S546L, l ebr fteKN aiyrqietepeec fthe of presence the require family KCNQ the of members All 2 . 6 ...] hs eut ugs htLT uain in mutations LQT1 that suggest results These s.e.m.)]. ora fCl cec 21)17 9335 doi:10.1242/jcs.147033 3943–3955 127, (2014) Science Cell of Journal n 6 5 o R562M, for 5 3, n P 5 , 3, uatP2Ta h itlKN1Ctriu,which C-terminus, KCNE1 impedes distal LQT5 the the of at one P127T and KCNQ1 mutant interaction in PIP2 mutants impairs that LQT1 C involving helix one KCNQ1 module: and C C-terminus helix distal at located KCNE1 mutations interacting LQT the of mechanisms pathways the different in two involved the summarizing Cartoon (C) i.8 Q1mttosi CQ ei show C PIP helix to KCNQ1 binding in impaired mutations LQT1 8. Fig. muolto PIP of immunoblot phosphorylation. o.()Qatfcto ftepl-onnraie to normalized pull-down ( the input of lower the Quantification in (B) shown row. are Inputs or row). C-terminus (upper KCNQ1 mutants LQT1 WT His-tagged of beads agarose .1 55% 0.01; P , .0;17% 0.001; n 5 I n ) * 3). KS 5 6 euainb oiomdae PKA yotiao-mediated by regulation 3, P 2 o R555C, for 6 , P o rprfnto Gme and (Gamper function proper for .5 ** 0.05; , 2 6 ulon(I2P)b PIP by PD) (PIP2 pulldown .0;Fg AB(l eut are results (all 8A,B Fig. 0.001; o K557E, for 6 P 2 , . I A Representative (A) .1 *** 0.01; KS n I 5 KS hne ucin On function. channel 3, KCNQ1–KCNE1 P P , , 2 n I 0.001. KS 5 idn n a and binding .5 34% 0.05; 3, urnsby currents P 2 , -coated 0.001 3951 6 I KS 7 2

Journal of Cell Science hwdta hslne sntrqie o PIP for study required recent not a is However, linker 2008). this al., that showed et (Hernandez networks ucinlywt PIP with functionally 56 a necpinaddslydasmlrsniiiyt Dr- to sensitivity WT similar as a displayed VSP and exception an was S546L lower significantly exhibited mutants LQT1 PIP to all binding that found lobe rpsdt eipratfrPIP for important be in to residues proposed KCNQ1, been have In 2013; also linkers 2003). segment-4–segment-5 al., al., and basic et et segment-2–segment-3 Zhang (Telezhkin the of 2011; 6 cluster al., segment et a the of Thomas both C-terminus, downstream in just proximal identified, residues KCNQ2 have Others and 2012). KCNQ1 al., et (Aivar KCNQ2 sdsge oachieve AC9, to and PDE4D3 designed PP1, PKA, is 2013), yotiao, with together al., KCNE1, and et located regions (Zaydman and 6. sites regions segment membrane downstream contact conserved the multiple including by molded Hradze l,20) hsbsccutrwssgetdto suggested was cluster subunits basic KCNQ2–KCNQ4 PIP This of with 2008). C-terminus interact al., the connecting in et linker B (Hernandez the and within A residues helix basic of cluster conserved euecM-nue CQ hshrlto tS7and S27 at to but phosphorylation interaction KCNQ1 KCNQ1–yotiao subsequent recently the has cAMP-induced 2009). 6 preserve al., reduce segment to et KCNQ1 Terrenoire shown in 2002; been A341V al., ways mutation et Marx LQT1 various al., 2012; The et in al., Kurokawa et 2005; disrupted Li Kass, 2004; and be (Chen conditions can disease under and stimulation adrenergic atrdatvto ieis uhfaue r ugsieof suggestive channel–PIP are state. features closed channel Such the of densities, and kinetics. stabilization relationship current deactivation conductance–voltage smaller with the faster of co-expressed significantly shifts C- mutants showed depolarizing LQT1 KCNE1 KCNE1, these with WT addition, interaction K557E, In R555H, physical (R555C, terminus. their LQT1 C the impaired helix study, present KCNQ1 R562M), the in in located Notably, 2005). mutations al., et Park 2010; 59,i neat ihcoetrl(oa ta. 2014). al., et (Coyan cholesterol with interacts it R539W, ifrn ein noe ihcutr fbscrsde have residues PIP basic with 2011). of interact al., to clusters et proposed Thomas with been 2005; endowed al., regions et Park Different 2003; al., et (Loussouarn ARTICLE RESEARCH 3952 the of question the raise PIP also multiple They of significance 8C). (Fig. modulation been have and C PIP helix residues for coiled-coil basic important addition, be in In to 2013). identified suggested al., been et Zaydman have 2010; al., et fD-S,mtnsR5C 55,K5EadR562M WT and to compared K557E decline R555H, current faster R555C, I significant activation a mutants Dr-VSP upon that exhibited phosphatase indicate Dr-VSP, voltage-dependent results Our the of 2009). of al., et use (Okamura the by and lse fbscrsde nKN1hlxCmgtb involved be might C helix KCNQ1 in PIP residues in basic of cluster eitdKN1popoyaina 2 u aldt upregulate to resulting cAMP- failed but the its S27 spared at C-terminus phosphorylation KCNQ1 KCNE1 mediated in D76N mutation LQT5 PIP bind not does C helix that possible is otiue lotrclyt ouaePIP modulate to allosterically contributes KS h ar-oeua cfodn ope omdb KCNQ1 by formed complex scaffolding macro-molecular The hs rprisaei iewt hs xetdfra impaired an for expected those with line in are properties These u eut ugs htKN1hlxCi rca o PIP for crucial is C helix KCNQ1 that suggest results Our hc ieyrfet hi oe fiiyfrPIP for affinity lower their reflects likely which , 2 ouain sn w opeetr prahs we approaches, complementary two Using modulation. I 2 KS I I KS KS neato,wihldu ohpteieta this that hypothesize to us led which interaction, 2 urn peuain(eja ta. 02.The 2012). al., et (Heijman upregulation current hnW smaue ypldw experiments pulldown by measured as WT than ugsigta tde o pert interact to appear not does it that suggesting , urn.I hssuy efudta h LQT5 the that found we study, this In current. 2 hog lcrsai n hydrogen-bonding and electrostatic through 2 oee,i spsil ht iemutant like that, possible is it However, . I KS 2 itrcigrgosi CQ.It KCNQ1. in regions -interacting hne euainfollowing regulation channel 2 enne ta. dniida identified al., et Hernandez . 2 neato Mtvle al., et (Matavel interaction 2 2 2 idn oapocket a to binding neato (Matavel interaction ietybtrather but directly 2 euainof regulation 2 Mutant . b 1- 2 CQ ei n h rxmt fKN1N n C-termini and with N- KCNQ1 interacting of C-terminus proximity the KCNE1 and C the helix KCNQ1 of location strategic Krkw ta. 03.A natKN1Ctriu has C-terminus KCNE1 of intact upregulation cAMP-dependent phosphorylation An for I crucial be KCNQ1 2003). to suggested is al., been PKA-mediated there KCNE1, et of of absence (Kurokawa the transduction 2003; in al., However, no et 2002). (Kurokawa al., expression et KCNE1 Marx for need the stimulation. without cAMP P127T following mutant LQT5 S92 the of of phosphorylation impact the the on examine to work future in hw ocuefntoa irpino cAMP-mediated of disruption functional previously cause the of mutation by a stimulation 2009), suppressed to al., is et motion shown (Haitin gated D76N this draw mutant that C-terminus LQT5 and KCNE1 together and C-terminus closer KCNQ1 the opening, fKN1adtesbeun hshrlto fS7 na during In that S27. found of also we phosphorylation study, subsequent FRET the N-terminus the dynamic and to C-termini yotiao- C-terminus KCNQ1 the the and for from of step N- PKA crucial a of KCNQ1 complex probably transfer is mediated channel This the 2009). the al., between C- et in (Haitin distance KCNE1 C-terminus the the KCNQ1 between shortens the interaction and have the we terminus that subunits, KCNQ1 labeled shown al., fluorescently the et previously and (Zheng between FRET C-terminus Using interaction KCNE1 2010). the direct Previous with unknown. a C-terminus proximal currently shown is have occurs studies transduction this which oepae yKN1i opigsympathetic coupling in KCNE1 by played role emnsb hwn htdlto fteKN1C-terminus KCNE1 the of deletion that ( showing by terminus hntpsadices hi ucpiiiyt ventricular to S27, susceptibility clinical Like severe conditions. their stress to As sympathetic increase individuals under tachyarrhythmias and 2004). affected and the S27 stimulation, al., phenotypes cAMP expose at upon et might phosphorylation increase they (Westenskow current KCNQ1 being prevent QTc impair in parents subsequently as longer mutations A341V and and his syncope both mutation 2000) than with LQT1 al., presented interval who the et interaction. proband (Splawski with another KCNQ1–yotiao both mutation associated described single the been additionally has a P127T preserving mutant as phosphorylation LQT5 while KCNQ1 the disrupt Interestingly, (Heijman to S27, KCNQ1 found KCNQ1 been in at has A341V impaired 2012) mutation 8C). to al., LQT1 (Fig. et one described stimulation leading only cAMP first date, mutant following To the S27 KCNE1 is at a phosphorylation P127T of mutation our example LQT5 To S27. the at P127T knowledge, phosphorylation P127T compromised mutant KCNQ1 that decreased the found we significantly for contrast, In KCNQ1– function. account channel impaired that mutant could an showed interaction nor We trafficking C. yotiao channel helix KCNQ1 defective impaired with neither mutation interaction P127T the physical addition, the In exposure. cAMP upon 9 a eetybe on ob hshrltdupon phosphorylated be to found been recently has S92 uatP2Ti CE itlCtriu e oasignificant a to led C-terminus basal distal in KCNE1 decrease in P127T mutant inln ops-hshrlto ucinleet ntecardiac the in I events functional post-phosphorylation to signaling hshrlto tS7adsbeunl rvn h cAMP- the prevent subsequently in and increase mediated S27 at phosphorylation deegcsiuainwe h hne slclzda the in at resulted event localized phosphorylation is this channel increased and the membrane, when plasma stimulation adrenergic KS KS D CQ a epopoyae tS7uo K activation, PKA upon S27 at phosphorylated be can KCNQ1 0–2)adteLT uainP2TdpesKCNQ1 depress P127T mutation LQT5 the and 109–129) hne.Hr,w apdteKN1efc oisdsa C- distal its to effect KCNE1 the mapped we Here, channel. urns(uoaae l,20) hc elcstecrucial the reflects which 2009), al., et (Kurokawa currents ora fCl cec 21)17 9335 doi:10.1242/jcs.147033 3943–3955 127, (2014) Science Cell of Journal I KS urn Lnb ta. 03.I ilb interesting be will It 2013). al., et (Lundby current I KS I KS urn Krkw ta. 03.Tu,the Thus, 2003). al., et (Kurokawa current urns hc eeual ob stimulated be to unable were which currents, I KS urns h oeua ehns by mechanism molecular The currents. b 1-adrenergic I KS channel b -

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DNA cloned were full constructs were All by KCNE1 respectively. verified vectors, and pEYFP-N1 and KCNQ1 pECFP-N1 mutagenesis were microscopy, into site-directed 109–129) TIRF Quikchange For and PCR-based (Stratagene). and (69–77 the (V109I deletions KCNE1 using C-terminus in introduced mutations KCNE1 LQT5 and the and P127T) R555C/ R562M) (S546L, allow and KCNQ1 to K557E in vector H, mutations pCDNA3 LQT1 into The cloned expression. were eukaryotic KCNE1 and KCNQ1 Human biology Molecular METHODS AND MATERIALS GX3 etr(hrai itc) h Q5mttoswere mutations the (Quikchange). LQT5 techniques into PCR The standard ligated by Biotech). were KCNE1, to (Pharmacia products introduced vector PCR The 2008). Digested pGEX-3X al., respectively. 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(Fig. modulation upregulation. current 9 n 3 and b -adrenergic 9 flanking 6 xnIsrmns,McootEcladPim50(GraphPad). 5.0 Prism and Excel ( Microsoft Conductance Instruments), Axon the cAMP, that with except at treatment above, was the the repolarization for For the as analyzed solution. were pipette relationships patch current–voltage the to added 200 PKA, o oprn Twt uatchannels. mutant with WT comparing for Student’s unpaired mean oln ih4 with boiling itdt igeBlzandsrbto codn to were according curves distribution Activation Boltzmann single conductance. a maximal to the fitted to normalized otg a tpe o from s relations, to 3 repolarization current–voltage for by followed the stepped increments establish was to voltage Then, stabilization. current afatvtdand half-activated efre sn osrds eoiae(R)cnuae anti-His (HRP)-conjugated PIP was For blots peroxidase ECL. western and boiled horseradish of antibody were Detection Eluates using buffer. glutathione). sample 20 performed and Laemmli mM, NaCl with in 150 (containing together 8, B pH buffer Tris-HCl using eluted 100 were complexes bound PMSF, lm ehiu speiul ecie Mie ta. 02.The 2012). al., et Na 5 (Meisel patch- KCl, 130 the described mM: of in previously contained configuration solution intracellular whole-cell as the technique in recorded clamp were cells CHO Electrophysiology o muorcpttos E 9 el eewse ihice-cold with washed were CaCl mM cells 1.5 (with 293 PBS HEK immunoprecipitations, For immunoprecipitations and assays Phosphorylation and serum calf fetal 10% 37 glutamine, at mM incubated 2 antibiotics, (DMEM) medium with Eagle’s 293) modified (HEK supplemented Dulbecco’s kidney in embryonic maintained human were and cells (CHO) ovary hamster Chinese transfections and culture Cell t600 at tro eprtr nbnigbfe otiig(nm) 10 mM): (in containing centrifugation buffer sample After binding Igepal. 0.25% 600 and in at NaCl 150 temperature 7.4, room pH HEPES at h 2 uat i-agdKN1Ctriu rtiswr nuae with incubated were PIP of proteins amounts C-terminus equal KCNQ1 His-tagged mutants soaiyt 9 Om h xenlslto otie i M:140 adjust MgCl mM): to (in 1.2 added contained KCl, solution was 4 external sucrose The NaCl, and mOsm. KOH), 290 with to osmolarity (adjusted 7.3 pH HEPES AEadfloe ywsenbotn.Iptsmlswr ae for taken ECL. were and HRP-conjugated antibody samples using anti-His performed Input was blotting. Detection western purposes. quantification by followed and PAGE Mrs.I xeiet novn APsiuain 0.5 stimulation, cAMP involving with experiments co-transfected In (2 1 were (Mirus). Dr-VSP and and of plate poly-L-lysine-coated (0.5 presence on multiwell KCNE1 seeded 24 and were a KCNQ1 cells in CHO coverslips 2012). glass al., et (Meisel ajse ihNO) n urs a de oajs soaiyto osmolarity to repolarization adjust by to followed at mV added held +30 were was Cells sucrose mOsm. and 320 NaOH), with (adjusted G elhae eeadd fe nuainudrrtto o at h 1 for rotation under incubation After 4 added. were Healthcare) (GE {1+exp[( iulzdb otaseto ihtepYPN etr(0.15 vector pEYFP-N1 the with were cells co-transfection Transfected by KCNE1. and visualized KCNQ1 with co-transfected also was F-agdW rmtn CQ n 2 and KCNQ1 1 mutant with transfected or and plate WT 1.5-mm multiwall CFP-tagged poly-L-lysine-coated 12-well a on in seeded coverslips were glass cells CHO imaging, TIRF el eesee n9-mdse n rnfce sn h calcium the using transfected and 5 dishes with 90-mm method on phosphate seeded 293 HEK were immunoprecipitation, and cells blotting western For KCNE1. mutant ˚ n he ahswt B otiig1 rtnX10ad1mM 1 and X-100 Triton 1% containing PBS with washes three and C aaaayi a efre sn h lmftporm(pClamp10, program Clampfit the using performed was analysis Data 6 g ...Saitclysgiiatdfeecswr sesdby assessed were differences significant Statistically s.e.m. g he ahsi idn ufrwr efre n olwdby followed and performed were buffer binding in washes three , ora fCl cec 21)17 9335 doi:10.1242/jcs.147033 3943–3955 127, (2014) Science Cell of Journal V n4 in 50 m 2 AP(im)pu 0.2 plus (Sigma) cAMP M 6 ˚ V .Pleswr rzni iudntoe n hnlysed then and nitrogen liquid in frozen were Pellets C. 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Journal of Cell Science ahnn . eae . uleae . ik . adnk,M n Romey, and M. Lazdunski, M., Fink, E., Guillemare, F., Lesage, J., Barhanin, o,P . aekr,C . egl .V,Hgis .L,Ai,E . Kennedy, J., E. Akin, L., J. Higgins, V., A. Weigel, J., C. J., Haberkorn, D., Mordel, P. Fox, J., Piron, Y., M. Amarouch, F., Abderemane-Ali, C., F. Coyan, Z., Es-Salah-Lamoureux, C., F. Coyan, F., Abderemane-Ali, S., F. Choveau, S. R. Kass, and L. Chen, Ferna P., Aivar, olce.Oem rti fec ellst a nuae ih5 with 4 incubated at was 500 lysate in Labs) cell (Alomone each antibody anti-KCNQ1 of protein mg One collected. apebfe,hae o i t55 at min 5 for heated buffer, sample ne oainfr1ha 4 at h 1 for rotation under ARTICLE RESEARCH 3954 and R. C. Burrow, L., M. Will, S., G. Jones, J., D. Tester, J., and J. M. L. Ackerman, Ptacek, C., M. Dalakas, S., Bendahhou, H., M. Butler, W., G. Abbott, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.147033/-/DC1 online available material Supplementary material Supplementary B.A.) (to and Research 1215/13]; Cardiovascular ISF number for [grant Fund Foundation Fields Science B.A, the Israel to the AT119/1-1 fund J.A.H.]: number and DIP [grant O.P. Projektkooperation Gorschungsgemeinschaft Deutsch-Israelische Deutsche the the by from supported is work This and Funding J.A.H. manuscript. work. the this wrote in B.A. help experiments. and the tools Y.H., designed invaluable data. B.A. provided the R.K. analyzed and and O.P. experiments the C.D., performed I.B.T.C. D.S., R.S., M.D., contributions Author interests. competing no declare authors The interests plasmids, Competing GPI-citrine and Dr-VSP the of gift kind (Tel respectively. their Hirschberg for Koret and Israel) Japan) University, University, Aviv (Osaka Okamura Yasushi thank We Acknowledgements olwdb he ahswt . lo Pbfe.Tefnlws was wash final The buffer. IP 30 in of resuspended then ml were Beads 0.5 water. distilled with with performed washes three 2500 at by centrifugation Sample followed temperature. room at incubated h and complexes 2 immune for the to added were (Pierce) beads agarose D-AEadfloe ywsenbotn.Frpopoyainof 37 phosphorylation For 250 with m blotting. treated were western cells HEK293 channels, by KCNQ1 followed and SDS-PAGE n niKN1popoS7(:5,Rbr .Ks,Uiest of University anti- laboratories). Kass, HRP-conjugated Jackson were (1:10,000, S. antibodies rabbit Secondary Robert York). (1:250, New Houston) Columbia, Center, (1:1000, phospho-S27 Science anti-KCNQ1 anti-yotiao Health (1:1000, Texas and Labs), of anti-KCNQ1 University Alomone were Dessauer, Carmen (1:200, antibodies anti-KCNE1 Primary Alomone), blotting. western and apebfe,dntrda 95 at denatured buffer, sample hncnrfgdfr2 i t10,000 at min were Lysates 20 (Sigma). for cocktail inhibitor centrifuged protease then Glycerol, and 10% EDTA, PMSF 10 1 NaCl, Triton, 150 1% 7.4, pH Hepes 50 mM): (in containing kdi cdi MM(ihu eu n niitc)fr1 i in min 10 for antibiotics) and serum (without DMEM in acid okadaic M oasu urn.(e comments). (see current. potassium G. nihdi otcledpamcrtclmfnto smmrn protein membrane as function reticulum M. M. endoplasmic Tamkun, hubs. cortical trafficking and in D. enriched Krapf, J., M. phosphatidylinositol-4,5- regulation. for channel cholesterol KCNQ1 substitutes in mutation bisphosphate QT Me long A M., (2014). Steenman, S., C. Nicolas, channels. other and KCNQ1/KCNE1 Pharmacol function: Front channel voltage-gated on PIP(2) Baro dance. different levels linker. protein A-B steady of helix control Kv7.2 specific the subunit by and expression Surface (2012). Rodrı mlctosfrgntcssetblt osde ada et n genetic and death cardiac sudden syndrome. QT to long congenital susceptibility for testing genetic for implications E. M. paralysis. Curran, periodic with associated is and Kv3.4 with A. S. Goldstein, ˚ .Clswr hnlsdwt Pbfe n yae eetetdwith treated were lysates and buffer IP with lysed then were Cells C. ˚ 19) ()Q1adlK(iK rtisascaet omteIK)cardiac I(Ks) the form to associate proteins (minK) lsK and K(V)LQT1 (1996). ne oain h olwn a,eulaonso protein-G– of amounts equal day, following The rotation. under C .adLusur,G. Loussouarn, and I. , ´ ge,M . iadz . iad,P,Aeo .adVlare,A. Villarroel, and P. Areso, P., Miranda, T., Giraldez, S., M. ´guez, ne-rh . oi-ee,C,Abri . lio A., Alaimo, A., Alberdi, C., Gomis-Perez, J., ´ndez-Orth, 20) tncdfeecsi ada oasu hne variants: channel potassium cardiac in differences Ethnic (2003). a.Cl Biol. Cell Nat. o.Bo.Cell Biol. Mol. 3 20) iP om oasu hnesi kltlmuscle skeletal in channels potassium forms MiRP2 (2001). 125. , 20) -iaeacoigpoen:dfeetpartners, different proteins: anchoring A-kinase (2005). ˚ n300 in C LSONE PLoS 21) poieefcso h 4S ikrand linker S4-S5 the of effects Opposite (2012). 7 ˚ 24 o i n ape eesbetdto subjected were samples and min 5 for C 1050-1051. , 2703-2713. , rt . aineu . hms .e al. et A. Thomas, C., Marionneau, J., ´rot, Nature m fimnpeiiain(P buffer (IP) immunoprecipitation of l g 21) lsammrn domains membrane Plasma (2013). aoCi.Proc. Clin. Mayo 7 ˚ n ujce oSDS-PAGE to subjected and C e47263. , n4 in 384 LSONE PLoS 78-80. , ˚ m n h uentn was supernatant the and C fI ufrfrovernight for buffer IP of l Cell 9 104 e93255. , m 78 CTpu 0.2 plus 8CPT M 217-231. , 1479-1487. , g o i was min 3 for m lof2 m gof 6 uoaa . hn .adKs,R S. R. Kass, and L. Chen, J., Kurokawa, atn . inr . hhm . eez . oe,E . hma,L., Shamgar, Spa J., B., Heijman, E. Cohen, A., Peretz, D., Shaham, R., Wiener, Y., Haitin, ebne .M n as .S. R. Kass, and M. J. Nerbonne, B. Y. Kubo, Attali, and and K. Nakajo, A. Peretz, M., Giladi, Y., Haitin, M., Dvir, E., Meisel, M. C. Lopes, and E. Medei, A., Matavel, rs,M,Hmod .R n il,B. Hille, and R. G. M. Hammond, D. M., Roden, Kruse, and S. Wells, T., Yang, S., Kupershmidt, H., Kanki, Hu J., Jr T. L., Jentsch, D. Minor, and M. S., J. Holton, A., Higashijima, K. Clark, M., J., R. Chahine, Howard, Y., Okochi, H., Iwasaki, I., M. Hossain, udy . nesn .N,Sefne,A . on . esrp .D., C. Kelstrup, H., Horn, B., A. Steffensen, N., M. Andersen, A., Lundby, Baro C., Bellocq, H., K. E., Park, Medei, G., I., Keselman, Loussouarn, L., J. Sui, A., Matavel, I., J. Remon, M., C. Lopes, W. C. Dessauer, and S. R. Kass, L., Chen, Y., Li, J. Cui, and B. Virgin-Downey, M., Guan, J., Shi, D., Wu, A., M. Zaydman, Y., Li, Kass, and T. Furukawa, L., Chen, A., Kaihara, R., J. Bankston, J., Kurokawa, S. R. Kass, and J. Rao, K., H. Motoike, J., Kurokawa, atn .adAtl,B. Attali, and Y. Haitin, S. G. Pitt, and A. D. Nunziato, S., Ghosh, S. M. Shapiro, and N. Gamper, kmr,Y,Mrt,Y n wsk,H. Iwasaki, and Y. Murata, Y., Okamura, R. A. Marks, J., D’Armiento, H., Motoike, S., Reiken, J., Kurokawa, O., S. Marx, enne,C . ak,O n hpr,M S. M. Shapiro, and O. Zaika, C., C. Hernandez, lbv .O n ihl,B J. B. Nichols, and O. O. Glebov, ak .H,Prn . aiee . Me S., Dahimene, J., Piron, H., K. Park, oto fcM-eedn K peuaini ua ogQ ydoetype syndrome long-QT G. human P. in Volders, upregulation and 1. IKs M. David, cAMP-dependent J., of L. control Windt, de R., I. subunits. channel potassium I(KS) of 28 motions gated and interactions Pongs,O.,Hirsch,J.A.andAttali,B. oiyKN hne function. channel protein. KCNQ modify KCNE1 of presence in the transitions and gating 34212-34224. sequential absence but the concerted undergo both not do channels interactions. KCNQ1 channel-PIP2 restoring by phenotype 1 (Austin) type QT long tutrlrqieetfrpoesn h ada +canlKCNQ1. channel K+ cardiac the processing Chem. for requirement structural dysfunction. by unravelled channelopathy. and voltage-sensing assembly 675. channel zebrafish (Kv7) KCNQ and into insight ascidian in sensor Y. voltage phosphatases. the Okamura, of and movement K. Nagayama, 21) nvv hshpoemc nlssrvastecrictresof targets cardiac V. J. the Olsen, reveals and signaling. analysis B. receptor M. phosphoproteomics adrenergic Thomsen, vivo N., Schmitt, In J., (2013). L. Jensen, C., Francavilla, homology channels. functional K+ rectifier a inward 5421. channels: and voltage-gated potassium between voltage-gated PIP2- KCNQ1/KCNE1 and D. E. regulation D. Escande, PLC-dependent Logothetis, modulates and channels. A K+ T. of kinase sensitivity Rohacs, Protein A., (2007). Rosenhouse-Dantsker, Y., 9 type Shen, heart. cyclase in adenylyl channel between potassium IKs formation the complex and facilitates Yotiao protein (PIP2) activity. channel 4,5-bisphosphate modulate 9095-9100. to phosphatidylinositol IKs of enhances sensitivity KCNE1 (2011). channel. potassium IKs the of (Austin) channel regulation PKA-dependent in potassium terminus heart S. a R. on Yotiao protein 16378. phosphorylation. anchoring PKA of A-kinase downstream the of channel. potassium heart USA a Sci. Acad. of Natl. regulation cAMP-mediated for expression PI(4,5)P2. by channels potassium module. 238-243. phosphoinositides. membrane repolarization. potassium KCNQ1-KCNE1 the of modulation receptor adrenergic channel. beta for S. R. Kass, and ei ikra h ieo hshtdlnstl45bshsht cino Kv7 on action 4,5-bisphosphate phosphatidylinositol of channels. site K+ the (M-type) as linker helix itiuindrn oaie ciaino h -elreceptor. T-cell with the interaction of activation disrupt localized that during distribution mutations syndrome long-QT by calmodulin. blocked is osoan G. Loussouarn, potentials. and actions 1994-2005. , ic Res. Circ. 20) CEvrat eelaciia oeo h easbntcarboxyl subunit beta the of role critical a reveal variants KCNE (2009). ora fCl cec 21)17 9335 doi:10.1242/jcs.147033 3943–3955 127, (2014) Science Cell of Journal 279 4 3 .Physiol. J. Science 3-11. , 16-24. , 33976-33983. , ic Res. Circ. 110 tes .L,Syn .R,Lnik . ujes .J,Boulet, J., H. Kuijpers, V., Lentink, R., S. 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