doi:10.1016/j.jmb.2008.06.010 J. Mol. Biol. (2008) 381, 569–580
Available online at www.sciencedirect.com
Inferred Motions of the S3a Helix duringVoltage-Dependent K+ Channel Gating
Anirban Banerjee and Roderick MacKinnon⁎
Laboratory of MolecularNeurobiology and Biophysics,Howard Hughes MedicalInstitute, Rockefeller University,Box 47, 1230 York Avenue,New York, NY 10065, USA
Received 7 April 2008;received in revised form30 May 2008;accepted 3 June 2008Available online10 June 2008
The gating of voltage-dependent potassium channels is controlled byconformational changes in voltage sensor domains. Previous studies haveshown that the S1 and the S2 helices of the voltage sensor are static withrespect to motion across the membrane, while the voltage sensor paddleconsisting of the C-terminal half of S3 (S3b) and the charge-bearing S4 ismobile. The mobile component is attached to S1 and S2 via the S2–S3 turnand the N-terminal half of S3 (S3a). In this study, we analyze KvAP, anarchaebacterial voltage-dependent potassium channel, to study the mobilitywith respect to translation across the membrane of S3a. We utilize an assaybased on attachment of tethered biotin and its site-specific accessibility toavidin. Our results reveal that the S3a helix does not move appreciablyacross the membrane in association with gating. The static behavior of S3aconstrains the conformations available to the voltage sensor when it closesand suggests that a set of negative countercharges within the membrane’sinner leaflet remains intact in the closed conformation.
Keywords: potassium channel; voltage-dependent gating; voltage sensor;membrane protein; electrical signaling
Edited by J. Bowie
Introduction
Cell membranes function as electrical capacitorscapable of storing a charge separation betweeninside and out. When a K+ channel opens, K+ flowsfrom inside the cell where the K+ concentration ishigher to the outside where the concentration islower. The net outward movement of K+ chargesthe membrane negative on its inner surface relativeto the outside. When Na+ channels open, theopposite occurs: Na+ flows inward from its higherconcentration outside, causing the inside to chargepositive. In this manner, ionic gradients andselective ion channels charge the cell membranecapacitor.1 The magnitude of the ionic gradientstypically found across living cell membranes givesrise to voltage differences of approximately 100 mV.Given the thickness of a cell membrane, approxi-mately 30 Å for the hydrophobic core, voltages of
this magnitude result in an electric field within themembrane greater than 107 V/m. In specialcircumstances, nature uses this strong electric fieldto control the conformation of membrane-embedded proteins to regulate their function. Adramatic example of this property is exhibited byvoltage sensors, which are membrane proteins thathave evolved specifically to undergo voltage-dependent conformational changes.2 Voltage sen-sors are best known as domains that regulate thegating of voltage-dependent ion channels.The atomic structures of voltage sensor domains
from two different voltage-dependent K+ (Kv)channels have been determined by X-ray crystal-lography. One of these is from a prokaryotic Kvchannel named KvAP and the other from aeukaryotic Kv channel, Kv1.2, and its variantnamed paddle chimera (Fig. 1).3–5 Despite theirdifferent origins, the two voltage sensor structuresare very similar. They consist of four mainly helicalmembrane crossings referred to as S1–S4. S4contains a basic amino acid (positively charged,usually arginine) every third amino acid. By exert-ing a force on these charges, the membrane electricfield performs electromechanical work to influencethe conformation of the voltage sensor.The paddle chimera structure provides a clear
picture of a voltage sensor in the context of a bilayer-
d.
Fig. 1 (legend on next page)
570 Voltage Sensor Motion during Gating of K+ Channels
571Voltage Sensor Motion during Gating of K+ Channels
like arrangement of lipid molecules.5 In this case, theK+ channel is open and the sensor is in a conforma-tion that brings positively charged amino acids onS4 near the extracellular membrane surface, wherewe expect to find them in an open-conformationvoltage sensor.6–8 The structure of a closed-confor-mation voltage sensor has not yet been determined;however, voltage sensor motions associated withmembrane voltage changes have been inferredusing state-dependent accessibility of cysteine resi-dues to thiol-reactive agents and state-dependentantibody binding.9,10
One accessibility method, which is to be themain method used in this study and is describedin detail below, employs avidin capture of biotintethered to specific sites on the channel (Fig.2a).9,11 Since avidin is a large macromolecule thatcannot gain access to relatively narrow aqueouscrevices in the protein, capture can occur only ifthe residue is positioned within a defined distance(depending on the tether length) from the mem-brane–water interface (Fig. 2b and c). Applied toKvAP, the biotin method showed that in associa-tion with gating, S1, S2, S5 and S6 remainrelatively static in the membrane with respect tomotion along a direction normal to the membraneplane (z-axis). By contrast, a segment of S4 appearsto change its position along the z-axis by around15 Å. These observations, taken together withcrystal structures showing the close apposition ofthe C-terminal half of S3 (S3b) and S4 (forming astructure called the voltage sensor paddle), suggestthat the voltage sensor paddle moves relative toS1, S2 and the pore (Fig. 1b and c).The region of KvAP corresponding to the S2–S3
turn and the N-terminal half of S3 (S3a) was notstudied using the biotin method. Structurally thisregion attaches the voltage sensor paddle to the S1–S2 half of the voltage sensor (Fig. 1b and c). It istherefore relevant to our understanding of voltagesensor motions to know whether S3a remains staticor moves with respect to the z-axis when thechannel gates. That is the question addressed in thisstudy.
Results
In the biotin–avidin assay, cysteine mutations areintroduced into a version of the channel in whichthe lone native cysteine was mutated to serine.Mutants were overexpressed, purified to homo-
Fig. 1. Residues tested in this study. (a) Sequence alignme5104624), rat Kv2.1 (GI, 24418849), rat Kv1.2 (GI, 1235594), Shastructure elements are indicated above the sequences. Selectedblue (positively charged) and green (hydrophobic). The residuechimera are highlighted in orange. (b) Stereo view of a subunProtein Data Bank (PDB) accession code 1ORS]. The α-carbon aspheres. (c) Stereo view showing a subunit of the paddle chimcarbon atoms of residues tested in this study have beenmappedgreen spheres.
geneity in the presence of detergents, and thenconjugated separately to cysteine-reactive linkersof various lengths with a biotin moiety attached totheir ends (see Fig. 2c for the chemical structuresof linkers used in this study). Conjugation wasconfirmed using a gel-shift assay in which thebiotin-conjugated channel was exposed to avidinand then run on non-reducing SDS-PAGE (Supple-mentary Fig. S1). The conjugated mutant channelswere reconstituted into lipid vesicles and thenincorporated into a planar bilayer system in whichthe voltage-gated ion conduction properties werestudied before and after the addition of avidin. Inseparate experiments, avidin was added to theintracellular or extracellular side of the membrane,defined with respect to the physiological orienta-tion of the channels in the membrane. Since thechannels undergo repeated opening and closingsteps during the assay, the measured accessibilityof biotin determines the cumulative accessibilityover all functionally relevant states sampledduring the open–closed transition of the channelmolecules. In all cases but one, the signature ofavidin binding is depletion of the current when themembrane is depolarized. In other words, whenwe observe a decrease in the current, we concludethat avidin has accessed the biotin. Inhibition isnot always complete and is frequently associatedwith changes in other characteristics of voltage-gated K+ conduction. However, for the purposes ofthis study, we are only interested in the binaryresult of accessibility or inaccessibility of the biotinmoiety to the added avidin. In control experi-ments, we have demonstrated that in one repre-sentative case, reduction of the disulfide linkageattaching the biotin tether to the cysteine residueresults in loss of susceptibility to avidin inhibitionof ion conduction. Also, in situ reduction of thedisulfide can reverse avidin inhibition of ionconduction. (Supplementary Fig. S2 and Supple-mentary Methods)In the two previous studies, residue positions in
S1, S2, S3b and S4 of the voltage sensor and S5and S6 of the pore were evaluated.9,11 Here wefocus on six positions shown as green spheresmapped onto KvAP (Fig. 1b) and the equivalentpositions of the paddle chimera channel (Fig. 1c).The intracellularmost position, A84C, conjugatedwith the 1.0 Å biotin-[2-(2-pyridyldithio)ethylamide](PDTE) linker, was inhibited by addition of intra-cellular avidin (Fig. 3a, left), but not when avidinwas added to the extracellular solution (Fig. 3a,
nt of the Kv1.2-2.1 chimera (paddle chimera), KvAP (GI,ker Kv (GI, 13432103) and KcsA (GI, 61226909). Secondary-conserved residues are shown in red (negatively charged),s tested in this study and the corresponding residues in theit of the isolated KvAP voltage sensor [grey ribbon trace,toms of the residues tested in this study are shown as greenera (grey ribbon trace, PDB accession code 2R9R). The α-onto the structure using the alignment in (a) and shown as
Fig. 2 (legend on next page)
572 Voltage Sensor Motion during Gating of K+ Channels
Fig. 3. Representative experi-ments showing the effect of avidinbinding to A84C KvAP conjugatedto tethers of different lengths. Cur-rent traces were elicited after depo-larization to positive voltagesbefore (black) or after the additionof internal (blue) or external (red)avidin. (a) 1.0 Å PDTE linker (n=3,internal; n=2, external). (b) 10 ÅIPEO linker (n=2, internal andexternal) and (c) 17 Å BCAC linker(n=3, internal; n=2, external). Cur-rent traces shown are average tracesof at least four traces.
573Voltage Sensor Motion during Gating of K+ Channels
right). Similar results were obtained with A84Cconjugated to the 10 Å linker (Fig. 3b). For the 17 Ålinker, avidin when added from either side (Fig. 3c,left) did not produce any measurable change in thevoltage-activated current. This is the first and onlycase in which we have observed the disappearanceof avidin’s effect with lengthening of a tether. Itseems possible that avidin binds in this case fromthe inside without influencing function because the
Fig. 2. Biotin–avidin capture assay. (a) Avidin being a largeand can only bind biotin when the tether length is sufficient tooutside of the membrane–aqueous border (right). (b) The porside. A biotin conjugated to a Cys residue in the protein can oCys lies within the effective tether length of the membrane–aqthis distance defines the zone of accessibility. (c) Detailed chemstudy - PDTE-biotin (biotin-[2-(2-pyridyldithio)ethylamide]),diamine), BCAC-biotin (biotinylcaproylaminocaproylaminoeand 7Å of the tether (shown in red) become buried. The remainshown on top of each tether.
17 Å linker is sufficiently long and located at theaqueous surface (biotin on a 1.0 Å tether is accessedhere) and outermost perimeter of the channel.Taken together, the data with different tetherlengths at position 84 place this site within 1.0 Åof the intracellular solution and greater than 17 Åfrom the extracellular solution. To reemphasize thepoint that the biotin assay determines the cumula-tive accessibility over all functionally relevant states
protein cannot penetrate into small aqueous crevices (left)allow the presentation of biotin beyond a defined distancee regions from two subunits of KvAP are shown from thenly bind avidin (shown as Cα trace in blue) if the Cα of theueous border (shown as black line). For the specific tether,ical structures of the tethered biotin reagents used in this
IPEO-biotin ((+)-biotinyl-iodoacetamidyl-3,6-dioxaoctane-thyl methanethiosulfonate). Upon avidin binding, biotinder of the tether defines the effective tether length, which is
574 Voltage Sensor Motion during Gating of K+ Channels
of the channel sampled during the open–closedtransition, these results indicate that position 84 iswithin 1.0 Å of the intracellular solution at sometime (if not all the time) and is greater than 17 Åaway from the extracellular solution all the time.Moving in the direction away from the intracel-
lular solution and in towards the membrane, wenext examined residue K88 (Fig. 1b). When functio-nalized with biotin on the 1.0 Å PDTE linker, avidininhibited channel activity from the intracellular side(Fig. 4a, left) but not from the extracellular side (Fig.4a, right). Both the 10 and the 17 Å linkers showedaccessibility to avidin from the intracellular side aswell (Fig. 4b, left, and c, left) but not from theextracellular side (Fig. 4b, right, and c, right). Thus,position 88 falls within the same region as position84, very near the intracellular solution.Based on the crystal structures, the next position,
L91, is expected to lie within the membrane’s
hydrophobic core. Consistent with this expectation,avidin from the intracellular solution failed to inhibitthe 1.0 Å conjugate, implying that the site is greaterthan 1.0 Å away from the intracellular solution (Fig.5a, left). Lengthening the tether to 17 Å permittedinhibition from the intracellular side (Fig. 5b, left).Both the 1.0 and 17 Å conjugates were inaccessiblefrom the extracellular solution (Fig. 5a and b, right).Measurements on L91C conjugatedwith the 10Å ((+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine)(IPEO) linker were complicated by a “run-down”phenomenon in which current slowly decreased overtime even in the absence of avidin when held at anegative holding voltage and repeatedly pulsed atfixed intervals to a constant positive voltage step. Anexample of this effect is shown in Fig. 5c. Even withthis background current decay, inhibition by avidinfrom the intracellular side was still discernable as animmediate decrease in the current following addition
Fig. 4. Representative experi-ments showing the effect of avidinbinding to K88CKvAP conjugated totethers of different lengths. Currenttraces were elicited after depolariza-tion to positive voltages before(black) or after the additionof internal(blue) or external (red) avidin. (a)1.0 Å PDTE linker (n=2, internal andexternal). (b) 10 Å IPEO linker (n=2,internal and external) and (c) 17 ÅBCAC linker (n=2, internal andexternal). Current traces shown areaverage traces of at least four traces.
Fig. 5. Representative experi-ments showing the effect of avidinbinding to L91C KvAP conjugatedto tethers of different lengths. Cur-rent traces were elicited after depo-larization to positive voltagesbefore (black) or after the additionof internal (blue) or external (red)avidin. (a) 1.0 Å PDTE linker (n=5,internal and external). (b) 17ÅBCAClinker (n=3, internal; n=2, external).Current traces shown are averagetraces of at least four traces. (c andd)L91C conjugated to the 10 Å IPEOlinker. (c) Individual current traceselicited by depolarization to thesame positive voltage step repeti-tively at the same fixed interval(left). Graph showing the meancurrent elicited at each pulsebetween fixed time intervals withinthe pulse as a function of time(right). No avidin was added(n=2). (d) Individual traces show-ing currents elicited after depolar-ization to the same positive voltagebefore (black) and after (blue) theaddition of avidin to the internalside (n=2).
575Voltage Sensor Motion during Gating of K+ Channels
of avidin (Fig. 5d). This result, together with datafrom the 1.0 and 17 Å conjugates, places position 91greater than 1.0 Å and less than 10 Å away from theintracellular solution and greater than 17 Å awayfrom the extracellular solution.The next positions evaluated—94, 96 and 98—
were extremely difficult to study due to a combina-tion of run-down phenomenon and few functionalchannels per membrane. Nevertheless, we were ableto obtain membranes reproducibly that contained at
least a few stable voltage-dependent channels. Thesemembranes yielded clearly interpretable data for asingle linker length on each of these three positions.Channels with the 10 Å IPEO linker conjugated atposition 94 were not affected by addition of avidinto either side of the membrane, suggesting that thisposition is greater than 10 Å from either membranesurface at all times during the gating transition(Fig. 6a). Channels with the 17 Å biotinylcaproylami-nocaproylaminoethyl methanethiosulfonate (BCAC)
Fig. 6. Representative experi-ments showing the effect of avidinbinding to different mutants ofKvAP conjugated to biotin linkers.Individual traces elicited afterdepolarizing to a constant positivevoltage step at fixed intervals areshown before (black) and afteraddition of internal (blue) or exter-nal (red) avidin. (a) I94C conjugatedto the 10 Å IPEO linker (n=2,internal and external). (b) A96Cconjugated to the 17 Å BCAC linker(n=3, internal and external). (c)V98C conjugated to the 10 Å IPEOlinker (n=3).
576 Voltage Sensor Motion during Gating of K+ Channels
linker conjugated at position 96 were inhibited byavidin from the intracellular solution (Fig. 6a, left)but not from the extracellular solution (Fig. 6b,right). Channels with the 10 Å IPEO linker con-jugated at position 98 were not inhibited from theextracellular solution (Fig. 6c). Because biotin con-jugated with the 10 Å linker to position 94 isinaccessible from the internal solution (Fig. 6a, left),internal avidin was not tested with this linker atposition 98.
Discussion
We have now scanned every membrane-spanningregion of the KvAP K+ channel using the biotin–avidin method. It is noteworthy that the S2–S3 turnand S3a helical segment is the least tolerant regionin terms of achieving analyzable voltage-dependentchannels with a tethered biotin. In other regions ofthe channel, conjugation of biotin influenced themidpoint of the Boltzmann activation curve and thekinetics of activation, deactivation, and inactivationto variable extents. However, it was still possible toclose and open the channels with voltage steps fromvery negative voltages (near −100 mV) to verypositive voltages (near 100 mV). In S3a not all tether
lengths could be studied, but the data are sufficientto address the question we set out to answer: doesthe S3a segment move on the z-axis with gating?Figure 7 shows the new data acquired in this study
mapped onto an atomic structure together with datafrom previous studies.5,9,11 The biotin accessibilitydata were all obtained using KvAP in planar lipidmembranes.9,11 The structural model is that of thepaddle chimera channel.5 The sequence alignmentcorrelating amino acid positions in KvAP and thepaddle chimera channel is given in Fig. 1a. Data arepresented for each tether length separately: blackspheres show positions inaccessible from either sideof the membrane while blue, red and yellow spheresshow positions accessible from the intracellular,extracellular, and both solutions, respectively. Planarsurfaces on the stereo images indicate the demarca-tions between accessible and inaccessible positionsas defined by the previously determined static (withrespect to the z-axis) helical components S1, S2, S5and S6. It is evident that the newly mapped sites inthe S2–S3 turn and S3a helix exhibit the sameaccessibility pattern as the static components. Wetherefore conclude that S3a does not undergosignificant z-axis translational movements whenthe voltage sensor undergoes its voltage-dependentconformational changes. This finding is consistent
Fig. 7. Summary of the biotin–avidin accessibility data. Stereo view of a single subunit of the paddle chimera withaccessibility data from the previous study11 and the current study mapped onto it using the alignment in Fig. 1a. Aminoacids are represented by their α-carbon atoms shown as a sphere. Red residues are accessible only to external avidin, blueresidues are accessible only to internal avidin, black residues are inaccessible, and yellow residues are accessible to avidinfrom both sides. Horizontal planes demarcate between regions of accessibility and inaccessibility. For the 17 Å BCAClinker, a single plane separates internal and external accessibilities.
577Voltage Sensor Motion during Gating of K+ Channels
with the idea that the S2–S3 turn at least in partserves as an anchor at the intracellular membrane–water interface.12 The sensitivity of this region of thechannel to perturbation (i.e. biotin conjugation)
implies that it might undergo some degree ofstructural rearrangement in association with gating,however, the motions appear not to be in thedirection of the z-axis.
578 Voltage Sensor Motion during Gating of K+ Channels
The conclusion that S3a does not change its depthwithin the membrane during gating has twoimplications for voltage sensor conformationalchanges. The first implication is that the large z-axis translation of S4 (see outlying accessibilitypattern on S4 in Fig. 7) does not come about throughslippage or translation of S3a relative to S1 and S2. Inthe absence of S3a z-axis motion, 15 Å translations ofS4 would require either slack in the loop connectingS3 to S4 or a break in the S3 helix so that S3b canmove when the channel gates. KvAP has no loopbetween S3 and S4 and experiments with the ShakerK+ channel show that it functions quite normallywhen the S3–S4 loop is deleted.13 Hence motion of
Fig. 8. Structural evidence for conformational changes of tthe voltage sensors of the isolated voltage sensor of KvAP (PDBvoltage sensor from the paddle chimera (PDB accession codedone using main-chain atoms of residues 90–97 in KvAP andvoltage sensor and part of the S4–S5 linker of paddle chimerastatic elements are shown in grey α-carbon trace and labeled inand labeled in red. Selected residues are shown in ball-and-st(mobile elements); grey, carbon (static elements); blue, nitroIonized hydrogen bonds between acidic and basic residues ar
S4 cannot be structurally accommodated merely byconformational change in the loop preceding S4.Certain aspects of the voltage sensor structure
support the idea of a break or hinge-like motionwithin S3, between S3a and S3b. First, sequencealignment of voltage-gated potassium channelsreveals a conserved proline residue in S3 thatdemarcates S3a and S3b. The proline, by disruptingthe normal hydrogen-bonding pattern of an α-helix,might serve to introduce a weak point, conservedthrough evolution, at which S3 can break underforce. Second, because S4 and S3 are physicallyjuxtaposed next to each other, even a minimalistmodel invoking large z-axis motion of S4 necessarily
he voltage sensor. (a) Superposition of the S3 and S4 fromaccession code 1ORS) shown in green ribbon trace and the2R9R) shown in cyan ribbon trace. The superposition wasresidues 256–263 in paddle chimera. (b) Stereo view of the(PDB accession code 2R9R). The view is from the side. Thegrey. The mobile elements are shown in red α-carbon traceick representation and are colored as follows: red, carbongen; red, oxygen; green, phenylalanine 233; cyan, water.e shown with dashed lines.
579Voltage Sensor Motion during Gating of K+ Channels
contradicts a completely static S3b. Third, in differ-ent structures of KvAP, S3b and S4 are associatedwith each other as a packed unit,3,12 suggesting thatthey move as a packed unit. For these reasons, wethink that the occurrence of a break in S3, betweenS3a and S3b, seems likely when the voltage sensorchanges from its open conformation (observed in thecrystal structure, Fig. 7) to its closed conformation. Acomparison of the crystal structures of the KvAPisolated voltage sensor and the paddle chimeravoltage sensor offer a suggestion as to how such abreak might occur during gating (Fig. 8a). In thepaddle chimera channel, the S3 helix axis is bentapproximately 30° at the junction between S3a andS3b. In KvAP, the helix is unwound at the junctionand S3b adopts a very different angle (than is seen inthe paddle chimera voltage sensor) with respect toS3a. The biotin accessibility of S3b suggests that itremains within the extracellular leaflet of themembrane bilayer even in the closed conformation(Fig. 7). A rotational motion centered about an axisnear the S3a–S3b demarcation would permit S3b toremain in the extracellular leaflet while enabling S4residues to move across the membrane and accountfor the pattern of biotin accessibility. The biotinaccessibility pattern together with the crystal struc-tures of paddle chimera and KvAP leads us to thegeneral conclusion depicted in Fig. 8b, which showsstatic elements of the voltage sensor (S1, S2 and S3a)in grey and mobile elements (S3b, S4 and the S4–S5linker) in red. Such motion of the S3b–S4 voltagesensor paddle could impart force to gate the pore viathe S4–S5 linker.The second implication of S3a maintaining a
constant depth within the membrane is that theenvironment created by S1, S2 and S3a observed inthe paddle chimera crystal structure (an openvoltage-dependent K+ channel) is likely to bepreserved in the closed conformation (Fig. 8b) ofthe channel.5 This environment provides acidicamino acids from S2 (E236), S3a (D259) and S0(E154), which form the internal negative cluster thatstabilize K302 and R305 in the open conformation.Preservation of this environment in the closedconformation would leave the internal negativecluster intact to stabilize the outermost S4 arginineresidues as they cross the phenylalanine gap to theinside of the membrane upon voltage sensorclosure.
Experimental Procedures
Preparation of biotinylated channels
The single native cysteine, C247, was mutated to C247Sto yield a version of KvAP without any cysteine. Cysteinemutations were introduced on this background using theQuikChange method (Stratagene) and the mutationsverified by sequencing through the entire gene. Mutantchannels were expressed, purified and functionalized withPDTE biotin, IPEO biotin or BCAC biotin as described.11,14
Biotinylated channels were reconstituted into lipid vesi-cles as described elsewhere.15
Electrophysiology
Electrophysiology of biotinylated channels was carriedout as described.14 Biotinylated channels were incorpo-rated into planar membranes and studied using variousvoltage protocols before or after the addition of 200 μg/mlavidin (Pierce) to one side of the membrane. For mostbiotinylated channels, the effect of avidin binding onchannel function was tested by 200-ms depolarizingpulses to +120 mV from a holding voltage of −120 mVevery 120 s. Some biotinylated channels, due to a shift involtage-dependent activation, required more negativeholding voltages (up to −160 mV).
Acknowledgements
We thank Seok-Yong Lee for the alignment in Fig. 1and Joel Butterwick for comments on the manu-script. This work was supported by NationalInstitutes of Health grant GM43949 to R.M. A.B. isa Damon Runyon Cancer Research Foundationpostdoctoral fellow and R.M. is an investigator inthe Howard Hughes Medical Institute.
Supplementary Data
Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2008.06.010
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