Cellular Biology - Attali lab...mia and sudden death in susceptible individuals.1–4 The stress reaction involves the release of stress hormones such as the glucocorticoid cortisol

Cellular Biology

Long QT Syndrome–Associated Mutations in KCNQ1 andKCNE1 Subunits Disrupt Normal Endosomal Recycling of

IKs ChannelsGuiscard Seebohm, Nathalie Strutz-Seebohm, Oana N. Ureche, Ulrike Henrion, Ravshan Baltaev,Andreas F. Mack, Ganna Korniychuk, Katja Steinke, Daniel Tapken, Arne Pfeufer, Stefan Kääb,

Cecilia Bucci, Bernard Attali, Jean Merot, Jeremy M. Tavare, Uta C. Hoppe,Michael C. Sanguinetti, Florian Lang

Abstract—Physical and emotional stress is accompanied by release of stress hormones such as the glucocorticoidcortisol. This hormone upregulates the serum- and glucocorticoid-inducible kinase (SGK)1, which in turnstimulates IKs, a slow delayed rectifier potassium current that mediates cardiac action potential repolarization.Mutations in IKs channel � (KCNQ1, KvLQT1, Kv7.1) or � (KCNE1, IsK, minK) subunits cause long QTsyndrome (LQTS), an inherited cardiac arrhythmia associated with increased risk of sudden death. Together withthe GTPases RAB5 and RAB11, SGK1 facilitates membrane recycling of KCNQ1 channels. Here, we show alteredSGK1-dependent regulation of LQTS-associated mutant IKs channels. Whereas some mutant KCNQ1 channels hadreduced basal activity but were still activated by SGK1, currents mediated by KCNQ1(Y111C) or KCNQ1(L114P)were paradoxically reduced by SGK1. Heteromeric channels coassembled of wild-type KCNQ1 and theLQTS-associated KCNE1(D76N) mutant were similarly downregulated by SGK1 because of a disruptedRAB11-dependent recycling. Mutagenesis experiments indicate that stimulation of IKs channels by SGK1 dependson residues H73, N75, D76, and P77 in KCNE1. Identification of the IKs recycling pathway and its modulation bystress-stimulated SGK1 provides novel mechanistic insight into potentially fatal cardiac arrhythmias triggered byphysical or psychological stress. (Circ Res. 2008;103:1451-1457.)

Key Words: kinase � trafficking � PIKfyve � LQT � stress

Physical and emotional stress may trigger cardiac arrhyth-mia and sudden death in susceptible individuals.1–4 Thestress reaction involves the release of stress hormones such asthe glucocorticoid cortisol via the hypothalamic–pituitary–adrenal axis.5 Cortisol regulates the expression of severalgenes, including the serum- and glucocorticoid-induciblekinase (SGK)16,7 that is abundant in cardiac tissue.8 Accord-ing to in vitro experiments SGK1 stimulates a slow delayedrectifier K� current (IKs)9 that mediates cardiac repolariza-tion. IKs is conducted by channels composed of KCNQ1 �subunits and KCNE1 � subunits.10,11 SGK1 phosphorylatesand thereby activates phosphoinositide 3-phosphate 5-kinase(PIKfyve), which generates PI(3,5)P2, which in turn enhancesRAB11-dependent insertion of KCNQ1/KCNE1 (Q1/E1)

channels into the plasma membrane.12 Accordingly, gain-of-function mutations of the genes encoding either SGK1or Q1/E1 are associated with shortening of the QT interval,an electrocardiographic measure of ventricular repolariza-tion time,13–15 whereas loss-of-function mutations lead toprolongation of the QT interval, causing long QT syn-drome (LQTS). Here, we study the ability of SGK1 torecover loss-of-function LQTS mutant channels and deter-mine the molecular requirements of SGK1 sensitivity.Stress-dependent stimulation of SGK1-mediated channelregulation might be of particular clinical importance forpatients with KCNQ1 or KCNE1 mutations who arepredisposed to potentially fatal cardiac arrhythmias trig-gered by physical and/or psychological stress.1,2

Original received August 14, 2007; resubmission received April 8, 2008; revised resubmission received October 9, 2008; accepted November 3, 2008.From the Department of Physiology I (G.S., N.S.-S., O.N.U., U.H., R.B., A.F.M., G.K., F.L.), University of Tuebingen, Germany; Department of

Biochemistry I (G.S., N.S.-S., U.H., K.S., D.T.), Receptor Biochemistry, Ruhr University Bochum, Germany; Institute of Human Genetics (A.P., S.K.),Technical University Munich, Germany; Institute of Human Genetics (A.P., S.K.), National Research Center of Environment and Health, Neuherberg,Germany; Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (C.B.), Università di Lecce, Italy; Department of Physiology andPharmacology (B.A.), Sackler Medical School, Tel Aviv University, Israel; INSERM U533 (J.M.), Institut du Thorax, Faculté de Médecine, Nantes,France; Department of Biochemistry (J.M.T.), School of Medical Sciences, University of Bristol, England; Department of Internal Medicine III (U.C.H.),Center for Molecular Medicine, University of Cologne, Germany; and Department of Physiology and Nora Eccles Harrison Cardiovascular Research &Training Institute (M.C.S.), University of Utah, Salt Lake City.

This manuscript was sent to Harry Fozzard, Consulting Editor, for review by expert referees, editorial decision, and final disposition.Correspondence to Prof Dr Guiscard Seebohm, Biochemistry I, Cation Channel Group, Room NC6/132, Ruhr University Bochum, Universitätsstr. 150,

D-44780 Bochum, Germany. E-mail [email protected]© 2008 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.108.177360

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http://circres.ahajournals.org

Materials and MethodsWestern Blot, Immunocytochemistry, andMolecular BiologyWestern blot of plasma membrane proteins and molecular biologywas performed as reported earlier.12 Cloning of RAB5, RAB7,RAB11, and FLAG-tagged KCNQ1 have been described previous-ly.16–19 Further details are available in the online data supplement athttp://circres.ahajournals.org.

ElectrophysiologyXenopus laevis oocytes were obtained according to German law asdescribed previously.12 Ovary lobes were digested with collagenase(type II; Worthington), and stage 5 oocytes were collected andinjected with 20 to 60 nL of cRNA. Oocytes were injected with 1 ngor 5 ng of KCNQ1 cRNA alone or with 1 ng KCNQ1 cRNA plus 1ng of KCNE1 cRNA or 5 ng SGK1, RAB5/7/11 cRNA. Oocyteswere stored for 3 to 4 days at 17°C in ND96 solution (in mmol/L: 96NaCl, 4 KCl, 1.8 MgC12, 1.0 CaC12, 5 HEPES; and 50 mg/Lgentamicin; pH 7.6). For voltage-clamp experiments the oocyteswere bathed in ND96 solution. A TurboTEC-10 amplifier (npielectronic, Tamm, Germany) was used to record currents at 24°C inoocytes 3 to 4 days after injection with cRNA using standard2-electrode voltage-clamp techniques. Data acquisition was per-formed using a Pentium IV computer, a Digidata 1322 A/D interface,and pClamp 8 software (Axon Instruments).

ResultsStructural Requirements for the Modulation of IKsChannels by SGK1Previous experiments from our laboratory indicated thatSGK1 enhances IKs by increasing the insertion of Q1/E1channels into the plasma membrane.12 The effect did notrequire the presence of KCNE1 � subunits (Figure 1a). Bycontrast, deletion of the N-terminal residues 1 to 81 inKCNQ1 (resulting in KCNQ1�N-term) completely abolishedthe stimulation by SGK1 (Figure 1b). To determine whethera specific region of KCNE1 modulates SGK1-sensitive recy-cling, we deleted its intracellular C terminus. Deletion of theC-terminal residues 73 to 129 within KCNE1 (resulting inKCNE1�C-term) abolished the stimulation of IKs by SGK1(Figure 1b). Thus, the enhanced plasma membrane insertionof Q1/E1 channels depends on the presence of both theKCNQ1 N terminus and the KCNE1 C terminus. Furthertruncations identified the 7-aa stretch from residues 73 to 79of KCNE1 as important for SGK1-dependent regulation ofQ1/E1 channels (Figure 1c). Cysteine-scanning mutagenesisof this region identified residues crucial for SGK1 activation.When H73, N75, or D76 were mutated to Cys, coexpressionof SGK1 had no effect or even reduced the current. Bycontrast, the P77C mutation facilitated the SGK1-mediatedstimulation of IKs (Figure 1c). These results demonstrate theimportance of the C-terminal HxNDP-containing region ofKCNE1 for targeted Q1/E1 vesicular transport to the plasmamembrane.

LQTS-Associated Mutations in KCNQ1 or KCNE1Can Disrupt SGK1-Dependent Modulation of IKsNext, we characterized the mechanism of SGK1-dependentmodulation of several LQTS-associated mutant IKs channels.Two common LQTS-associated missense mutations inKCNE1 are located within the 73 to 79 region, namely S74Land D76N.20 To characterize the mechanism of SGK1-

dependent modulation of these LQTS-associated mutant IKschannels we studied heteromeric channels coassembled ofKCNQ1 and KCNE1(S74L) (Q1/S74L) or KCNE1(D76N)(Q1/D76N) subunits. Q1/S74L channels were activated bySGK1 (Figure 5), whereas currents mediated by Q1/D76Nchannels were reduced by SGK1 (Figure 2a). Changes inplasma membrane-associated KCNQ1 protein suggestedthat this functional reduction in mutant IKs was caused bya trafficking defect. SGK1 increased wild-type Q1/E1 butnot Q1/D76N channel abundance in the plasma membrane,as assayed by Western blot and chemiluminescence anal-ysis of surface protein (Figure 2b and 2c).12 Furthermore,a chemiluminescence assay of oocytes injected withcRNAs encoding Myc-tagged KCNQ1 and KCNE1(D76N)showed that constitutively active SGK1(S422D) but not

Figure 1. Deletion of the KCNQ1 N terminus and disruption of amotif in KCNE1 impair SGK1-dependent stimulation. a, KCNQ1(Q1) channels are stimulated when coexpressed with SGK1 inoocytes. Channels were activated by 7-second pulses to 60 mV.Example traces are shown overlaid. Horizontal scale bar, 1 sec-ond; vertical scale bar, 1 �A. KCNQ1/KCNE1 (Q1/E1) coexpressedwith SGK1 yielded larger currents than Q1/E1 expressed alone inXenopus oocytes. Horizontal scale bar, 2 seconds; verticalscale bar, 3 �A. b, Deletion of the KCNQ1 residues 1 to 81(KCNQ1�N-term) and deletion of KCNE1 residues 73 to 129(KCNE1�C-term) render IKs channels insensitive to SGK1 (n�8 to18). To determine the potentiation by SGK1, current amplitudes inthe presence and absence of SGK1 were analyzed at the end of7-second pulses to 60 mV and the ratio was calculated. Drawingsindicate structure of channel subunits expressed. c, Deletion ofresidues 73 to 79 but not of residues 80 to 129 of KCNE1 abol-ishes stimulation by SGK1 at 60 mV. The residues 73 to 80 wereindividually mutated to Cys, and the resulting mutant channelswere tested for stimulation by SGK1 as described in b. A uniquemotif (HxNDP) was required for the SGK1 effects (n�7 to 15). TheKCNE1 constructs are illustrated on the left, and the numbers indi-cate deleted residues or the position of individual Cys substitu-tions. d, KCNQ1 and KCNE1 may interact at different sites witheach other, and correct interaction at these sites is a prerequi-site for stimulation by SGK1. Another requirement for this stimu-lation is intact Ser27, a target of PKA phosphorylation (supple-mental Figure I). Binding of �-tubulin to the KCNQ1 N terminus(possibly influenced by Ser27 and allowed by correct KCNQ1-KCNE1 interaction) may be a molecular linker to the cytoskele-ton and may allow specific and efficient sorting of KCNQ1 pro-teins into early endosomes.

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inactive SGK1(K127N) decreased KCNQ1 protein in theplasma membrane (Figure 2c). In COS-7 cells cotrans-fected with Q1/D76N and SGK1(S422D), no increase inplasma membrane immunofluorescent staining of KCNQ1was observed (Figure 2d). By contrast, we previouslyreported that constitutively active RAB11 increasedplasma membrane abundance of heterologously expressedwild-type KCNQ1 protein in COS-7 cells.12

The small G proteins RAB5 and RAB11 are expressed incardiac tissue and oocytes, where they constitute centralcomponents of recycling specificity and efficiency for vesi-cles containing wild-type IKs channels.12,20 RAB5 has beenimplicated in the regulation of early steps in the endocyticpathway, whereas RAB11 is localized at the trans-Golginetwork, post-Golgi vesicles, and the recycling endosome.21

Hydrolysis of GTP activates RAB-dependent vesicle traffick-ing.16,22–26 The IKs recycling pathway can be assayed by

injecting GTP into oocytes where RAB-dependent pathwaysare functionally impaired by overexpression of mutant formsof RAB5 and RAB11.12 Here, we used a similar approach toassay for the recycling pathway of mutant IKs channels.Oocytes expressing Q1/D76N channels were microinjectedwith GTP during voltage clamp, and ionic currents wererecorded. Q1/D76N-mediated currents were increased byGTP when channels were coexpressed with dominant-negative RAB11(S25N) or the switch2 domain mutantRAB11(T77A).27 However, no change in Q1/D76N-mediatedcurrents was noted when channels were coexpressed withGTP-insensitive RAB5(N133I) alone or in combination witheither of the RAB11 mutants (Figure 3a). Wild-type IKschannels colocalize with RAB1112; however, using greenfluorescent protein (GFP)-tagged KCNQ1 and DsRed-taggedRAB11 constructs, we observed no colocalization of RAB11with Q1/D76N channels in COS-7 cells (Figure 3c). Taken

Figure 2. SGK1 decreasesQ1/D76N current density byreducing plasma membraneabundance of the channels. a,KCNQ1 was coexpressed withKCNE1 carrying the LQTS-associated mutation D76N,which is localized in the regionimportant for SGK1 effects.Coexpression of the mutantchannels with SGK1 resulted indecreased currents. Channelswere activated by 7-secondpulses to varying potentials (�80to 60 mV in 20 mV steps; n�17to 20; horizontal scale bar, 1second; vertical scale bar, 3 �A).b, Biotinylation Western blotrevealed an increase of plasmamembrane KCNQ1 protein bySGK1 for wild-type Q1/E1 and adecrease for Q1/D76N. FourWestern blots were densito-metrically analyzed using Scionimage software. c, Chemilumi-nescence assay of KCNQ1(Myc-tagged between S1 andS2) coexpressed with wild-typeKCNE1 (E1) or KCNE1(D76N)(D76N) in the absence or pres-ence of constitutively activeSGK1(SD) or inactive SGK1(KN)mutant kinases. Data for wild-type KCNE1 are depicted inblack; data for KCNE1(D76N) arein gray. d, KCNQ1(FLAG)/D76Nwas coexpressed with a GFP-tagged constitutively activemutant SGK1(S422D) in COS-7cells (SGK1-expressing cells aregreen). KCNQ1(FLAG) wasprobed by immunostaining withan anti-FLAG antibody (red).Visual observation and 2D pixel-intensity analysis using ImageJsoftware suggest that theSGK1(S422D) mutant does not

markedly increase plasma membrane expression of KCNQ1(FLAG)/D76N channels (position and direction of analyzed areas are indi-cated by yellow arrows). The lower graph shows control data from KCNQ1(FLAG)/KCNE1(wt) channels (replotted from our previousstudy12). Here, the increased fluorescence in the plasma membrane can be observed. Error bars indicate �SEM. *Significant differ-ences (P�0.05).

Seebohm et al Disrupted IKs Trafficking in LQTS 1453



together, these results indicate that Q1/D76N channels areendocytosed by RAB5 and reinserted into the plasma mem-brane by a RAB11-independent mechanism.

RAB7 is a protein that has been implicated in the regula-tion of late endosomal steps in the endocytic and lysosomalpathways.21 To understand where Q1/D76N channels accu-mulate in the cell, we coexpressed mutant channels withRAB7 and the dominant-negative RAB7 mutant T22N.RAB7(T22N) increased only Q1/D76N-mediated currentsbut not wild-type IKs (Figure 4a). According to chemilumi-nescence, coexpression of Q1/D76N channels withRAB7(T22N) increased the amount of Q1/D76N plasmamembrane protein (Figure 4b). Cotransfection of wild-typeand mutant Q1/E1 channels with RAB7(T22N) in COS-7cells showed that Q1/D76N but not wild-type channelscolocalize with RAB7-positive late endosomal vesicles (Fig-ure 4c). These data suggest a close relationship of RAB7 withQ1/D76N but not wild-type channels.

To determine whether SGK1 modulates channels harbor-ing a LQTS-associated mutation, we examined 8 previously

characterized KCNQ1 mutants and the S74L mutant ofKCNE1. Six of the 9 mutant channels were stimulated bycoexpression with SGK1 (Figure 5). By contrast,Q1(P117L)/E1 channels were insensitive, and Q1(Y111C)/E1- and Q1(L114P)/E1-mediated currents were reduced bySGK1. These mutations were recently reported to disruptnormal trafficking.28 Interestingly, Q1(L114P) expressedwithout E1 was downregulated on SGK1 coexpression aswell, suggesting that E1 is not required for the inversed SGK1sensitivity (Figure I in the online data supplement). LikeQ1/E1(D76N) channels, Q1(L114P)/E1 channels were mod-ulated by RAB7(T22N) (Figure 4 and supplemental Figure II)and were mistargeted in transfected cardiomyocytes (supple-mental Figure III).28 Furthermore, Q1(Y111C)/E1 andQ1(L114P)/E1 channels colocalized with RAB7 but not withRAB7(T22N) in COS7 cells (supplemental Figure IV). Sim-ilar to KCNE1, the KCNQ1 residues required for activationby SGK1 are located in an N-terminal juxtamembranousregion.28 This raises the possibility that these regions ofKCNQ1 and KCNE1 interact to promote trafficking of theheteromeric channel complex (Figure 5, inset).

DiscussionIn a previous study, we showed that SGK1 enhances theinsertion of Q1/E1 channels into the plasma membrane.12

However, structural prerequisites have not been studied untilnow. The N terminus of KCNQ1 contains the importanttrafficking motif LEL and a critical tyrosine residue (Tyr51),both of which are required for Q1/E1 channel trafficking tobasolateral membranes in MDCK cells.29 The LEL motif, aswell as Tyr51, might be involved in the RAB5/11-dependentand SGK1-sensitive targeted recycling of IKs channels.12

Furthermore, the N terminus of KCNQ1 contains a PXXPsequence that may facilitate interactions with SH3 domains.Recently, direct interaction of the N terminus of KCNQ1 with�-tubulin in transfected COS-7 cells and in guinea pigcardiomyocytes was reported.30 Both the interaction with�-tubulin and the phosphorylation of Ser27 in KCNQ1 areprerequisites for protein kinase (PK)A-mediated activation ofthe channels.30,31 Interestingly, mutation of Ser27 or deletionof the N-terminal residues 1 to 81 in KCNQ1 (resulting inKCNQ1�N-term) disrupt its stimulation by SGK1 (Figure 1band supplemental Figure V). These data raise the possibilitythat stimulations by SGK1 or PKA require both the integrityof a macromolecular complex and an intact interaction withthe cytoskeleton.12,30,31 Misfolding of the � subunit KCNE1as a result of deletion of the intracellular domain or of specificsingle amino acid substitutions might disturb the integrity ofa macromolecular complex and/or cytoskeleton–KCNQ1 in-teractions, disrupting correct intracellular sorting to vesiclesthat are subject to SGK1-stimulated exocytosis. However,stimulation by SGK1 involves increased trafficking to theplasma membrane, whereas PKA-mediated stimulation ofKCNQ1 seems not to be related to trafficking events.12,30

Here, we show that stimulation by SGK1 does not require thepresence of KCNE1 � subunits (Figure 1a). However, whenKCNE1 is present, a short stretch (73 to 79 region) within theKCNE1 intracellular C terminus is required for stimulationby SGK1 (Figure 1b and 1c). Within this region, we identi-

Figure 3. Q1/E1(D76N) is endocytosed by a RAB5-dependentand recycled back to the plasma membrane by a RAB11-independent SGK1-sensitive pathway. a, Q1/D76N expressedalone, with GTP binding–insufficient RAB5(N133I), withdominant-negative RAB11(S25N), with switch2 domain mutantRAB11(T77A), or with combinations of the constructs. Oocytesexpressing Q1/D76N were injected with 0.23 nmol GTP throughglass pipettes while currents were recorded continuously by2-electrode voltage clamp (see inlay to the right). Injection ofGTP increased Q1/D76N-mediated currents when RAB11(S25N)or RAB11(T77A) were present (filled symbols). In oocytesexpressing Q1/D76N together with dominant-negativeRAB5(N133I) alone or in combinations with RAB11(S25N) orRAB11(T77A) (open symbols), GTP had no effect on Q1/D76Ncurrent amplitudes (n�3 to 8). These results indicate that inhibi-tion of RAB5(N133I) may block accumulation of Q1/D76N at theplasma membrane by RAB11-uncoupled exocytosis. b, GFP-tagged Q1/D76N (green) was coexpressed with DsRed-fusedRAB11 (red) or RAB11(S25N) (red) in COS-7 cells. Visual obser-vation and 2D pixel-intensity analysis using ImageJ software didnot suggest colocalization of the mutant channels with RAB11or RAB11(S25N) [position and direction of analyzed areas areindicated by yellow arrows, results of Q1/D76N scans are repre-sented as green curves, results of RAB11/RAB11(S25N) scansas red curves]. Error bars indicate �SEM.




fied 4 residues (H73, N75, D76, and P77) that are critical forthe normal effect of SGK1 on Q1/E1 channels (Figure 1c).These results indicate that the C-terminal juxtamembranousHxNDP-containing region of KCNE1 is important for tar-geted Q1/E1 vesicular transport to the plasma membrane. Theintact intracellular KCNE1 C terminus was shown to interactwith the sarcomeric protein T-cap, suggesting a T-tubule–myofibril linking system.32 Thus, Q1/E1 channel complexescontain several molecular components allowing for physicallinkage to cytoskeletal compartments, which may allowspecific and efficient trafficking along the cytoskeleton (Fig-ure 1d).

Two common LQTS-associated missense mutations inKCNE1 are located within the 73 to 79 region, namely S74Land D76N.33 Q1/S74L channels were activated by SGK1(Figure 5), whereas Q1/D76N-mediated currents were re-duced by active SGK1 (Figure 2a) possibly as a result ofreduced plasma membrane-associated KCNQ1 protein asdemonstrated by Western blot and chemiluminescence anal-ysis of surface protein (Figure 2b and 2c).12 By analysis of 9previously characterized LQT1 mutants, we identified 6mutant channels that were stimulated by coexpression withSGK1 (Figure 5). By contrast, 3 mutant channels were eitherinsensitive or inhibited by SGK1. These 3 mutations (P117L,Y111C, and L114P in KCNQ1) were recently reported todisrupt normal trafficking.28 We previously reported that con-stitutively active SGK1 increased plasma membrane abundanceof heterologously expressed wild-type KCNQ1 protein inCOS-7 cells.12 This effect is absent in COS-7 cells cotransfected

with Q1/D76N and constitutively active SGK1(S422D) (Figure2d). Thus, the SGK1-stimulated plasma membrane insertionof Q1/E1 is disrupted in several LQT1 mutant channels and 1LQT5 mutant Q1/E1 channel. Similar to KCNE1, theKCNQ1 residues required for activation by SGK1 are locatedin an N-terminal juxtamembranous region.28 This raises thepossibility that these regions of KCNQ1 and KCNE1 interactto promote trafficking of the heteromeric channel complex(Figure 5, inset).

RAB5 has been implicated in the regulation of early stepsin the endocytic pathway, whereas RAB11 is localized at thetrans-Golgi network, post-Golgi vesicles, and the recyclingendosome.21 The D76N mutation in KCNE1 uncouples IKschannels from normal RAB11-dependent endosome recy-cling to the plasma membrane and induces a distinct, RAB11-independent recycling pathway (Figure 3a). On the contrary,wild-type IKs channels colocalize with RAB11.12 Takentogether, these results indicate that Q1/D76N channels areendocytosed by RAB5 and reinserted into the plasma mem-brane by a RAB11-independent mechanism. The lack ofacute functional effects of RAB11(S25N) or RAB11(T77A)(Figure 3a) and the lack of colocalization with RAB11(Figure 3b) suggest that RAB11-dependent vesicle recyclingto the plasma membrane might be disrupted in Q1/D76Nchannels. Consequently, Q1/D76N channels may escapeRAB11-dependent recycling, and formation of storage vesi-cles (as was observed for wild-type IKs channels) may becompromised.

RAB7 is a protein implicated in the regulation of lateendosomal steps in the endocytic and lysosomal pathways.21

Figure 4. RAB7 modulates cur-rent density and plasma mem-brane abundance ofQ1/E1(D76N) channels. a, Q1/E1and Q1/D76N were expressed inoocytes in the absence or pres-ence of either wild-type RAB7 orthe dominant-negative mutantRAB7(T22N). Q1/E1 currents andQ1/D76N currents were analyzedat the end of a 7-second pulseto 60 mV and normalized to theQ1/E1 current and Q1/D76N cur-rent, respectively (n�12 to 47).Data are represented asmeans�SEM. b, Oocytesexpressing KCNQ1-Myc/E1 orKCNQ1-Myc/D76N were injectedwith RAB7 cRNA or RAB7(T22N)cRNA. After 3 days, plasmamembrane expression of Myc-tagged protein was analyzed bya chemiluminescence assay. Theresults were normalized to theQ1/E1 and Q1/D76N values,respectively. Data are represent-ed as means�SEM. c, GFP-tagged Q1/E1 and Q1/D76Nwere coexpressed with DsRed-

fused RAB7 or RAB7(T22N) in COS-7 cells. Q1/E1 channels were expressed intracellularly and in the plasma membrane and did notcolocalize with intracellularly expressed RAB7 or RAB7(T22N). However, GFP-tagged Q1/D76N colocalized to some degree withDsRed-fused RAB7 but not with RAB7(T22N), as suggested by visual observation and 2D pixel-intensity analysis using ImageJ soft-ware. The position and direction of analyzed areas are indicated by yellow arrows, results of GFP-Q1/E1 and GFP-Q1/D76N scans arerepresented as green curves, and results of DsRed-RAB7 and DsRed-RAB7(T22N) scans as red curves. Interestingly, colocalizationwas mostly detected in intracellular compartments. Error bars indicate �SEM, and significant differences (P�0.05) are marked by anasterisk (*).




The dominant-negative RAB7 mutant T22N increased onlyQ1/D76N-mediated currents but not wild-type IKs (Figure 4a)by increasing the amount of Q1/D76N plasma membraneprotein (Figure 4b). Like Q1/E1(D76N) channels,Q1(L114P)/E1 channels were modulated by RAB7(T22N)(Figure 4 and supplemental Figure II). Furthermore, cotrans-fection of wild-type and mutant Q1/E1 channels withRAB7(T22N) in COS-7 cells showed that Q1/D76N but notwild-type channels colocalize with RAB7-positive late endo-somal vesicles (Figure 4c). Furthermore, Q1(Y111C)/E1 andQ1(L114P)/E1 channels colocalize with RAB7 but not withRAB7(T22N) in COS7 cells (supplemental Figure IV). Threefindings suggest that the disease-associated mutant channelsmay be stored in late endosomes and possibly the endoplas-mic reticulum (ER): (1) Q1/D76N channels do not colocalizewith RAB11 and can be trafficked back to the plasmamembrane by a GTP-dependent but RAB11-independentprocess (Figure 3a and 3c); (2) Q1/D76N channels [andQ1(Y111C)/E1 and Q1(L114P)/E1 channels] colocalize in anintracellular compartment with RAB7 and are modulated byRAB7(T22N) (Figure 4); and (3) SGK1 treatment does notresult in additional fractional bands in Western blots, indicat-ing that Q1/D76N channels are not trafficked to lysosomesand digested by enzymes.

SGK1-mediated phosphorylation of PIKfyve and subse-quent PI(3,5)P2 production act to regulate channel activity viaRAB11-dependent vesicle exocytosis (Figure 6). Taken to-gether, our findings suggest that Q1/D76N, Q1(Y111C)/E1,and Q1(L114P)/E1 channels are trafficked forward to RAB7-dependent late endosomal vesicles and/or the endoplasmicreticulum. Indeed, Q1(Y111C)/E1 and Q1(L114P)/E1 were

reported to be enriched in the ER.28 The localization ofKCNE1(D76N) seems to be altered in stem cell–derivedventricular myocytes as well (supplemental Figure V). Analtered localization of Q1(Y111C)/E1, Q1(L114P)/E1, andQ1(P117L)/E1 channels has been reported for cardiac myo-cytes.28 Stimulation of RAB11-dependent exocytosis willresult in increased endocytosis of plasma membrane contain-ing mutant Q1/E1 channels, reducing channel density in theplasma membrane and thereby current amplitudes (Figure 6).Mutant channels stored in late endosomes, ER, and possiblyGolgi apparatus can potentially be trafficked to the plasmamembrane, and stimulation of this ER export forward traf-ficking by GTP would explain the RAB11-independent stim-ulation of Q1/D76N-mediated currents (Figure 3a).

The present findings bear potential clinical significance.Carriers of the E1(D76N), Q1(Y111C), or Q1(L114P) muta-tions may benefit from avoidance of situations (eg, sustainedstress, dexamethasone treatment, and excessive blood insulinlevels) that might stimulate SGK1 and lead to an even greaterdecrease in IKs and prolongation of QT intervals.

In summary, our studies demonstrate a link between alteredvesicle recycling of disease-associated mutant IKs channelsand the stress-dependent kinase SGK1.

Sources of FundingThis work was supported by the Deutsche Forschungsgemeinschaft(La315/4-5 and SFB), a stipend from the Gottlieb Daimler-und KarlBenz-Stiftung (to G.S.), a stipend from the Erwin-Riesch-Stiftung (toG.S.) and MIUR-PRIN2004 (to C.B.), and a Deutsche Forschungs-gemeinschaft stipend (GRK 1302/1) (to U.H.). S.K. was funded by

Figure 5. Differential SGK1 sensitivity of Q1/E1 channels con-taining LQTS1-associated mutations. Several LQTS-associatedmutations in Q1 reduced IKs in oocytes by 40% to 70% com-pared to wild-type Q1/E1. SGK1 partially recovered function ofmost mutant channels (n�7 to 20). The pulse protocol used isdescribed in Figure 1. Error bars indicate �SEM (*P�0.05).Inset, Approximate positions of LQTS-associated point muta-tions studied here are indicated (circles). Location of mutationsleading to SGK1-mediated reduction in IKs are shown as lightgray [Q1(Y111C), Q1(L114P)] or dark gray [KCNE1(D76N)] filledcircles.

Figure 6. Diagram of Q1/E1 and Q1/D76N channel recycling.Wild-type Q1/E1 channels are endocytosed by a RAB5-dependent mechanism and reinserted/recycled by a RAB11-dependent mechanism. RAB11-dependent Q1/E1 exocytosis isenhanced by SGK1, an effect involving the phosphorylation andactivation of PIKfyve and the generation of PI(3,5)P2. This mech-anism is disrupted in Q1/D76N channels. Q1/D76N channels aresimilarly endocytosed via a RAB5-dependent endocytosis butare forward-trafficked to RAB7-enclosing late endosomal vesi-cles and possibly the ER and Golgi apparatus. Stimulation ofRAB11-dependent exocytosis leads to increased membrane fluxinto the plasma membrane, resulting in increased endocytosis.Because KCNE1(D76N)-containing channels are not enriched inRAB11 vesicles, their exocytosis is not stimulated, but they areendocytosed, resulting in reduced functional expression of thesedisease-associated channels. However, injection of GTP stimu-lates trafficking of Q1/D76N channels from the late endosomesand the ER or Golgi apparatus independent of RAB11 function.




German National Genome Research Network grant 01GS0838 andthe Leducq Fondation.

DisclosuresNone.

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CIRCRESAHA/2008/177360/R2

1Su

Supplement Material Online Figure I SGK1 inhibits Q1(L114P) currents in the absence of KCNE1.

Large amounts of cRNA (12 ng per oocyte) encoding the LQTS-associated mutant KCNQ1(L114P) were injected into Xenopus laevis oocytes. Coexpression of these mutant channels with SGK1 (5 ng of cRNA per oocyte) resulted in decreased currents measured at 60 mV (KCNQ1(L114P), n = 12-16). Data are represented as mean + SEM.

Online Figure II Mutant Q1/E1 channels are modulated by RAB7(T22N).

Heteromeric channels composed of KCNE1 and either the Y111C or the L114P mutant of KCNQ1 were expressed in oocytes in the absence or presence of RAB7 or RAB7(T22N). Q1/E1-current amplitudes were determined at the end of a 7-s pulse to 60 mV and normalized to the respective amplitudes in the absence of RAB7 (n = 23-29). Data are represented as mean + SEM.




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Online Figure III Localization of KCNE1-EGFP and KCNE1(D76N)-EGFP in stem cell-derived murine cardiac myocytes.

EGFP-tagged wild-type KCNE1 or KCNE1(D76N) were transfected into stem cell-derived murine ventricular myocytes, and the green EGFP fluorescence was detected by confocal microscopy. Three representative examples of each transfection are shown. KCNE1-EGFP seemed to be relatively evenly distributed in the cells, whereas KCNE1(D76N)-EGFP localized to an unidentified fibrous structure, as suggested by visual observation and 2D-pixel intensity analysis using ImageJ-software. The position and direction of analysed areas are indicated by yellow bars, results of scans are presented. Analysis of frequencies of pixel intensity peaks revealed an increase in narrow peaks. These peaks indicate an enrichment of EGFP-tagged KCNE1(D76N) and to a lesser extent of EGFP-tagged KCNE1wt to intracellular fibre-like structures. Data are represented as mean ± SEM.




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Online Figure IV Colocalisation of RAB7 and LQTS-associated mutant Q1/E1 channels.

VSV-tagged mutant Q1/E1 concatemers as described by Dahimene et al. 1 were coexpressed in COS-7 cells with DsRed-fused RAB7 or RAB7(T22N). VSV-tagged mutant Q1/E1 concatemers partially colocalized with DsRed-fused RAB7 but not with RAB7(T22N), as suggested by visual observation and 2D-pixel intensity analysis using ImageJ-software. The position and direction of analyzed areas are indicated by yellow bars, results of VSV-tagged mutant-Q1/E1 concatemer scans are represented as green curves, results of DsRed-RAB7 and DsRed-RAB7(T22N) scans as red curves. Interestingly, the colocalization is mostly seen in restricted intracellular compartments.




4

Online Figure V SGK1 inhibits function of phosphorylation-deficient mutant Q1(S27A/D)/E1 channels.

Ser27 in KCNE1 (orange circle) is located in the N-terminal region and is subject to PKA-mediated phosphorylation 2. Q1/E1 channels were expressed in oocytes in the absence or presence of SGK1. Amplitudes of Q1/E1- and Q1(S27A/D)/E1-mediated currents were determined at the end of a 7-s pulse to 60 mV and normalized to the respective amplitudes in the absence of SGK1 (n = 8-11). Data are represented as mean + SEM.

Methods:

Stem cell-derived ventricular cardiac myocytes - Cor.Ve murine ventricular cardiomyocytes

(CellSystems Biotechnologie Vertrieb GmbH, St. Katharinen, Germany) are derived from

transgenic mouse embryonic stem cells. These cells are a set of ESC-derived 99.9% pure

ventricular cardiomyocytes that exhibit normal morphology and physiological behavior. They

can be used for electrophysiology (patch clamp), cardiotoxicity, and other functional studies

(http://www.axiogenesis.com/cms/front_content.php?client=1&lang=1&idcat=3). The cells

possess puromycin resistance and GFP reporter genes driven by a cardiac-specific promoter

(rlc2v promoter) and are obtained by in vitro differentiation and puromycin selection of

mouse ES cells. The cells were thawed, seeded onto fibronectin-coated dishes, and then

incubated for 48 h for complete recovery. Subsequently, transfection of Cor.Ve ventricular

myocytes with KCNE1-EGFP or KCNE1(D76N)-EGFP was performed by Fugene 6

transfection according to the manufacturer’s protocol. Fluorescence was detected using a




5

confocal microscope (LSM 510, Zeiss) with adequate filter sets. The settings of confocal

imaging (filter set, detection intensity) of all images were identical.

Western Blot – Western blot of plasma membrane proteins was performed as reported earlier

3. Intact healthy oocytes were incubated in 1 mg/ml Sulfo-NHS-LC-Biotin (Pierce, USA) for

30 min at room temperature. After washing five times in ND96, 20 intact oocytes were

homogenized in 400 µl H-buffer (in mM: 100 NaCl, 20 Tris-HCl, pH 7.4, 1% Triton X-100,

plus a mixture of protease inhibitors, CompleteTM, Roche, Germany) and kept for 1 h at 4°C

on a rotator. Thereafter, the lysed oocytes were centrifuged for 1 min at 16,000 x g. The

supernatants were supplemented with 25 µl NeutrAvidin Biotin-Binding Protein (Pierce,

USA) and incubated for 3 h at 4°C on a rotator. The beads were then pelleted by

centrifugation for 2 min at 1600 x g and washed three times in H-buffer. The pellets were

boiled in 40 µl SDS-PAGE loading buffer (sodium dodecyl sulfate-polyacrylamide gel

electrophoresis, 0.8 M 2-mercaptoethanol, 6% SDS, 20% glycerol, 25 mM Tris-HCl, pH 6.8,

0.1% bromophenol blue). Finally, the samples were Western-blotted and probed with primary

KCNQ1 antibody (anti-KCNQ1, 1:100 dilution, Santa Cruz: sc-10646) and secondary

antibody (anti-rabbit, Santa-Cruz).

Molecular Biology – The molecular biological procedures were the same as previously

described 3. Human KCNQ1 and SGK1 were subcloned into oocyte expression vectors psp64,

a modified pcDNA3.1 vector, or pSGEM. The clones were mutated at the positions

mentioned in the text by site-directed mutagenesis using PCR with cloned Pfu-polymerase

(Invitrogen, Germany). Cloning procedures of wt and mutant RAB5, RAB7, RAB11, and

FLAG-tagged KCNQ1 have been described previously 4-7. SGK1(S422D) and SGK1(K127N)

were subcloned into pIRES2-EGFP. All constructs were confirmed by sequencing. In vitro




6

synthesis of cRNA was performed with SP6 and T7 mMessage mMachine kits (Ambion via

Applied Biosystems, Germany).

Immunocytochemistry – COS-7 cells were grown on glass coverslips and fixed with 4%

paraformaldehyde 3 days after transfection. Cells were subsequently stained with an anti-

FLAG antibody (anti-FLAG polyclonal antibody from rabbit, F7425, Sigma, Germany) to

detect the FLAG-tagged KCNQ1 as described before 3. Immunostaining of VSV-tagged

mutant Q1/E1 concatemers was performed as described by Dahimene et al. 2 using the same

anti-VSV antibody (1:500 dilution, Sigma). Fluorescence was detected using a confocal

microscope (LSM 510, Carl Zeiss, Germany) with adequate filter sets.

Chemiluminescence assay – Experiments were performed as described previously 3. Oocytes

expressing KCNQ1 with an extracellular myc-tag (between S1 and S2) were incubated for

30 min in ND96 with 1% bovine serum albumin (BSA) at 4°C. Oocytes were subsequently

incubated with rat monoclonal anti-myc antibody (Roche, 100 µg/ml, dilution: 1:100 in ND96

+ 1% BSA) for 1 h at 4°C, washed 5 times at 4°C with ND96 + 1% BSA, and incubated with

2 µg/ml peroxidase-conjugated affinity-purified F(ab)2 fragment goat anti-rat IgG antibody

(Jackson ImmunoResearch, England) in ND96 + 1% BSA for 1 h. Oocytes were washed

thoroughly for 5 min at 4°C with ND96 + 1% BSA and then 5 times for 5 min at 4°C with

ND96. Individual oocytes were transferred to 100 µl Power Signal Elisa solution (Pierce,

USA), and chemiluminescence was measured with a multilabel counter (Wallac Victor,

Perkin Elmer, Germany). The results from 20 oocytes were averaged and are presented in

relative light units (RLU).




7

Online data reference List:

1. Dahimène S, Alcoléa S, Naud P, Jourdon P, Escande D, Brasseur R, Thomas A, Baró

I, Mérot J. The N-terminal juxtamembranous domain of KCNQ1 is critical for channel

surface expression: implications in the Romano-Ward LQT1 syndrome. Circ Res.

2006;99:1076-83.

2. Marx SO, Kurokawa J, Reiken S, Motoike H, D'Armiento J, Marks AR, Kass RS.

Requirement of a macromolecular signaling complex for beta adrenergic receptor

modulation of the KCNQ1-KCNE1 potassium channel. Science. 2002;295:496-

9.

3. Seebohm G, Strutz-Seebohm N, Birkin R, Dell G, Bucci C, Spinosa MR, Baltaev R,

Mack AF, Korniychuk G, Choudhury A, Marks D, Pagano RE, Attali B, Pfeufer A,

Kass RS, Sanguinetti MC, Tavare JM, Lang F. Regulation of endocytic recycling of

KCNQ1/KCNE1 potassium channels. Circ Res. 2007;100:686-692.

4. Hoekstra D, Tyteca D, Van IJzendoorn SC. The subapical compartment: a traffic

center in membrane polarity development. J Cell Sci. 2004;117:2183-2192.

5. Choudhury A, Dominguez M, Puri V, Sharma DK, Narita K, Wheatley CL, Marks

DL, Pagano RE. Rab proteins mediate Golgi transport of caveola-internalized

glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells. J Clin Invest.

2002;109:1541-1550.

6. Sharma DK, Choudhury A, Singh RD, Wheatley CL, Marks DL, Pagano RE.

Glycosphingolipids internalized via caveolar-related endocytosis rapidly merge with

the clathrin pathway in early endosomes and form microdomains for recycling. J Biol

Chem. 2003;278:7564-7572.

7. Kanki H, Kupershmidt S, Yang T, Wells S, Roden DM. A structural requirement for

processing the cardiac K+ channel KCNQ1. J Biol Chem. 2004;279:33976-33983.



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