Proc. Natl. Acad. Sci. USAVol. 93, pp. 11652-11657, October 1996Cell Biology

Single residue substitutions that change the gating properties of amechanosensitive channel in Escherichia coliPAUL BLOUNT*, SERGEI I. SUKHAREV*, MATrHEW J. SCHROEDER*, SCOTr K. NAGLE*t, AND CHING KUNG*t*Laboratory of Molecular Biology and tDepartment of Genetics, University of Wisconsin, 1525 Linden Drive, Madison, WI 53706

Communicated by William A. Catterall, University of Washington School of Medicine, Seattle, WA, July 30, 1996 (received for review May 2, 1996)

ABSTRACT MscL is a channel that opens a large pore inthe Escherichia coli cytoplasmic membrane in response tomechanical stress. Previously, we highly enriched the MscLprotein by using patch clamp as a functional assay and clonedthe corresponding gene. The predicted protein contains alargely hydrophobic core spanning two-thirds of the moleculeand a more hydrophilic carboxyl terminal tail. Because MscLhad no homology to characterized proteins, it was impossibleto predict functional regions of the protein by simple inspec-tion. Here, by mutagenesis, we have searched for functionallyimportant regions of this molecule. We show that a shortdeletion from the amino terminus (3 amino acids), and alarger deletion of 27 amino acids from the carboxyl terminusof this protein, had little ifany effect in channel properties. Wehave thus narrowed the search of the core mechanosensitivemechanism to 106 residues of this 136-amino acid protein. Incontrast, single residue substitutions ofa lysine in the putativefirst transmembrane domain or a glutamine in the periplas-mic loop caused pronounced shifts in the mechano-sensitivitycurves and/or large changes in the kinetics of channel gating,suggesting that the conformational structure in these regionsis critical for normal mechanosensitive channel gating.

have recently been shown to gate in response to osmoticchanges as assayed in whole-cell (protoplast) patch clamp (13).The MS-channel activities observed in E. coli have been

shown to survive detergent extraction, biochemical fraction-ation, and reconstitution into artificial liposomes (6, 7, 14).Using patch clamp as a functional assay, we previously bio-chemically enriched the MscL protein. These studies led us tothe protein's amino-terminal sequence, and we ultimatelycloned and sequenced the corresponding gene, mscL. Theexpression of mscL has been shown to be both necessary andsufficient for MscL-channel activity (7, 15). As part of thisevidence, it has been shown that a mscL-null strain of bacteriastill displayed MscS, but not MscL, activity, whereas reintroduc-tion of mscL via an expression plasmid restored wild-type MscLactivity (7). Here we use this native expression system to studyengineered mutants of MscL. By generating deletions and site-directed mutations, we narrowed the search for functionallyimportant regions of the protein to two hydrophobic domains,their flanking regions, and a periplasmic loop; we have also foundtwo residues involved in determining channel kinetics and settingthe threshold for mechanoactivation.

Mechanosensation is a necessary primary event for many senses,from hearing and maintaining balance to cardiovascular andosmotic regulation. Electrophysiological evidence has suggestedthat one of the primary ways mechanical force may be detectedis through mechanosensitive (MS) ion channels (1, 2). AlthoughMS channel activity is apparently important for many functionsand has been found in a variety of cell types (1), the molecularparticipants and mechanisms are only now being discovered.Perhaps one of the major reasons that the biochemical andmolecular properties of MS channels have not been extensivelycharacterized in eukaryotes is that, unlike ligand- or voltage-gated channels, MS channels are typically found in low abundancein tissues or cells that are difficult to obtain.

Bacteria can serve as a model for the study of the basicprinciples of mechanosensory transduction in cells. Previousstudies have shown that when patches excised from Escherichiacoli giant spheroplasts are stressed by a pressure gradient atleast two channel activities are observed: one with fast kineticsand a very large conductance of about 2.5 nS (called MscL formechanosensitive channel of large conductance), and an ac-tivity with slower kinetics and a smaller conductance of -0.8nS (MscS) (3-7). MS-channel activities have been observedand examined in several other bacterial species (8-12). Thesebacterial MS channels have been implicated in sensing and/oradapting to changes in the osmotic environment. For example,both MS-channel activities and the efflux of small osmolytes inresponse to osmotic shock were found to be blocked withsimilar concentrations of gadolinium, a nonspecific blocker ofvarious types of MS channels (10). Also, MS channels in E. coli

MATERIALS AND METHODS

Strains and Expression Constructs. Derivatives of the E.coli strain AW405 (16) were used to host MscL expressionconstructs. PB104 is PB1l1 bacteria (7) bearing a completesubstitution of a chloramphenicol cassette for the mscL gene.Briefly, the entire mscL open reading frame was deleted froma PstI genomic clone by mutagenesis (described below) usingthe oligonucleotide TGG TCG GCT TCA TAG GCC TCCCAG TTC CCC CG. The StuI site generated from thismutagenesis was used to insert the chloramphenicol fl cas-sette. The PstI fragment was then used for the generation of thenull mutant as described (7). A nonsuppresser E. coli strain,AW737 (17), was used to generate PB106 as performed forPB101 (7). No differences were seen in the expression levels ofmutants in the PB101, PB104, or PB106 strains. The cloningvector pBluescript (Stratagene) was used for most intermedi-ate cloning steps, and pBlOa plasmid (7) was used as theexpression vector. Controls used throughout the manuscriptinclude the parental strain (AW405), and PB104 carrying themscL expression plasmid p5-2-2 (7) (referred to here as"expressed wild type"). Expression of all mutants was per-formed in the MscL null mutants described above. All strainswere grown at370C in Luria-Bertani medium (LB); ampicillin(100 ,ug/ml) was added for strains containing plasmids. Forinduction of the pBlOa-based plasmids, cultures were grown tomid-logarithmic phase (OD600, 0.5). Isopropyl ,B-D-thiogalac-topyranoside. (IPTG; 1 mM) (Sigma) was then added andbacteria were grown for an additional 2 h.

Abbreviations: MS, mechanosensitive; IPTG, isopropyl ,B-D-thiogalactopyranoside.tPresent address: Pritzker School of Medicine, University of Chicago,Chicago, IL 60637.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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Molecular Modifications of the mscL Gene. The carboxyl-terminal deletion mutants were generated by polymerase chainreaction (PCR) (18) using primers that inserted the stop codonTAA in the position indicated, truncated the DNA to besubcloned, and added an additional XhoI site for subcloning.For the generation of the other mutations, the insert fromplasmid p5-2-2 (7), bearing the entire MscL open readingframe, was subcloned into phage M13 mpl8 from pBluescriptas a KpnI-HindIII fragment. The amino-terminal deletionsand site-directed mutageneses were performed either by theBurke-Olson technique (19) with modifications (20), or usingthe Sculpture IVM kit (Amersham).

Single-Channel Analysis. Spheroplasts were generated andused in patch-clamp experiments (21) as described (3, 22). Thepipette solution was 200 mM KCl, 90 mM MgCl2, 10 mMCaCl2, and 5 mM Hepes adjusted to the pH indicated in Table1; the bath solution was the same plus 0.3 M added sucrose. Allrecordings were performed at -20 mV at room temperature;we have shown the inward current of channel openings as

upward. Records were analyzed with PCLAMP6 software. Pres-

sure sensitivity was assessed using a pressure transducer (Mi-cro Switch; Omega Engineering, Stamford, CT) calibrated bya pneumatic transducer tester (Bio-Tec, Winooski, VT) and ispresented normalized to MscS (applied pressure/thresholdpressure required to open MscS in the same patch). BecauseMscS desensitized rapidly under the conditions used, it was notpossible to determine the mid-pressure at which 50% of MscSin the patch were open. We therefore used the threshold ofMscS opening to approximate a point of reference for eachpatch. MscS threshold was determined as the pressure needed,within 7 sec, to open two or more channels as evidenced bytwo-storied events. The MscL threshold was defined as thepressure at which openings were readily observed every 0.5 to2 sec; this corresponded to a measured PO of approximately0.0002 to 0.003 (depending upon the kinetics of the mutantsand assuming -100 channels in the average patch). Althoughthe absolute thresholds of MscS and MscL should depend on

the number of channels in the patch, we routinely observedthat the latter was 1.4 times the former in all wild-type patches.This ratio was far more constant than the absolute pressures

Table 1. Analysis of channel open times and relative threshold sensitivities

Open time, ms Pressure sensitivity

MscL expressed Codon T2 T3 (relative to MscS)pH 6.0

Parental AW405 6 ± 1 37 ± 7 1.42 ± 0.03Expressed wild type 8 ± 1 28 ± 2 1.42 ± 0.03K31C TGC 6 ± 1 27 ± 3 1.45 ± 0.03K31R AGG 6 ± 1 44 ± 5 1.46 ± 0.03K31E GAG 1.0 ± 0.1* 3.2 ± 0.1* 1.20 ± 0.04*K311 ATT ND ND NDQ56P CCT 7 ± 1 27 ± 4 0.85 ± 0.02*Q56C TGC 13 ± 3 160 ± 30* 1.45 ± 0.03Q56F TTT 27 ± 3* 216 ± 38* 1.52 ± 0.03Q56W TGG 24 ± 10 174 ±39* 1.52 ± 0.04Q56Y TAC 62 ± 6* 399 ± 46* 1.49 ± 0.02Q56L TTA 4 ± 1 16 ± 2 1.53 ± 0.03Q56R CGT 5 ± 1 17 ± 4 1.21 ± 0.04*AQ56 NC NC 2.20 ± 0.05*A&2-4 4±1 21±2 1.49±0.02A2-12 ND ND NDA110-136 7 ± 1 30 ± 4 1.46 ± 0.03A104-136 ND ND ND

pH 5.0Expressed wild type 4 ± 1 17 ± 3 1.38 ± 0.03Q56H CAT 3.6 ± 0.3 12 ± 1 1.19 ± 0.03*

pH 7.5Expressed wild type 4 ± 1 23 ± 6 1.39 ± 0.04Q56H CAT 34 ± 7* 222 ± 42* 1.46 ± 0.03Data from an E. coli wild-type strain, and either mscL genes of wild type ("expressed wild type") or

mutants expressed in a MscL-null-mutant strain are presented. Mutants are labeled as the wild-type aminoacid, the residue number, and the mutant amino acid; the codons, subsequent to mutagenesis, are shownfor the point mutations. Deletions are indicated with a A followed by the amino acids deleted. All kineticdata were derived from at least six independent experiments (obtained from at least two independentspheroplast preparations) each containing at least 125 events that were fit to a three-open state model;T2 and T3 are the two longer time constants. The shortest T for each group, which was always <0.3 ms,could not be measured accurately and is not shown (but see Fig. 2). Mean ± SEM for all other values areshown. All groups had a minimum of 2000 total events for analysis. Analysis of an individual patch atdifferent pressure stimulation demonstrated that channel mean open time did not vary significantly overthe range of PO used for these experiments. For example, in one experiment using a patch containingapproximately 100 channels, at P0 of 0.0006, T2 = 9.5, T3 = 25.8; at PO 0.0075, T2 = 10.0, T3 = 23.0. ND,not determined because MscL activity was never seen (n 6), even when patches from these groups werestimulated with pressures .2 times that required to open MscS. NC, not calculated because MscL activitywas observed in only five patches prior to patch breakage; the few channel openings observed werehundreds of ms in duration.*P 0.001 as determined by Student's t test. Each group was compared to the expressed wild type at the samepH; the pH was the same in the pipette and in the bath for the data tabulated. When the expressed wild-typewas compared at different pH values, no significant difference was seen. The definitions of MscS and MscLthreshold and the reason for using the threshold ratio to approximate the ratio of the gating forces aredescribed in Materials and Methods. The 1.4 pressure ratio was observed at all pH values measured.

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required for channel activities in different patches. No signif-icant difference in the pressure required to open MscS (150 ±

80 mmHg; mean ± SD) was observed between the expressedwild type analyzed at pH 6.0 and any other group. Data were

acquired with a 3-kHz filtration, a sampling rate of 5 kHz, andfurther filtering in PCLAMP6 Fetchan at 1 kHz.Antibody Generation, Western Blots, and PhoA Assays.

Antibodies were raised against a MscL carboxyl-terminalpeptide: CEIRDLLKEQNNRS (Chiron). The peptide was

coupled to keyhole limpet hemocyanine (Sigma) activated by1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochlo-ride according to the protocol provided by Pierce. Rabbitswere immunized according to standard procedure (23) and thepostimmune serum was taken in 24-day intervals. Antibodieswere screened using Western blot procedure against totalmembrane proteins from AW405, expressed wild type, andPB104. For the Western blot shown, proteins were fractionatedin 12% SDS/polyacrylamide minigels and electroblotted to an

Immobilon P membrane (Millipore). A ProtoBlot Westernblot AP kit (Promega) was used.

RESULTS

MS Channel Activity Is Encoded Within the First Four-fifths of the MscL Protein. Since nothing had been previouslyknown concerning the molecular basis of mechanosensitivity,and because the MscL protein is relatively small, it was difficultto predict, a priori, whether the entire MscL protein was

required for mechanosensing and ion conduction. Hence, we

have performed deletions at both termini of MscL followed byanalysis of channel function. Deletion of only 3 (A2-4), but not11 (A2-12) amino acids from the amino terminus was toleratedto produce a functional MscL channel. On the other hand,deletion of up to 27 amino acids at the carboxyl terminus(mutant A110-136) left the channel functional (Table 1).However, when an additional 6 amino acids (RKKEEP, notethe clustered charged residues) were deleted from the carboxylterminus, no channel activity was detected (A104-136; see

Table 1). Although the lack of activity in the A104-136 mutantis difficult to interpret because we cannot assay for proteinexpression using the antipeptide antibody to the portion deleted(see below), the observation that the A110-136 mutant doesfunction shows that most of the hydrophilic carboxyl-terminal endplays no major role in the channel properties assayed. Theseobservations are in stark contrast to the finding that single residuesubstitutions elsewhere in MscL can dramatically change channelkinetics and/or sensitivity (see below).

Single Residue Substitutions Suggest Functional Regions ofMscL. Mutations were engineered at selected hydrophilicresidues in this highly hydrophobic protein to test the impor-tance of these regions in the normal function of the MscLchannel (Table 1). We selected two residues for this analysis;one is a charged residue within the first hydrophobic domain,and the other is a hydrophilic residue within a more hydrophilicregion in the center of the molecule. Absolute MS channelpressure sensitivity was observed to be variable betweenpatches, presumably because of differences in patch size,geometry, or cell-wall digestion. We therefore used MscS as an

internal standard to assess the pressure sensitivity of thewild-type and mutated MscL channels. When recorded at -20mV, wild-type MscL channels were consistently found to open

at -1.4 times the pressure required for opening MscS (Table1, Fig. 1A Upper, and ref. 6). Several single residue changesaltered this threshold sensitivity of the MscL channel: deletionof glutamine 56 (AQ56) decreased the sensitivity of thechannel, whereas several substitutions of this residue (Q56R,Q56P, and Q56H at pH 5.0), and a mutation of K31 to E,

increased its sensitivity (Table 1). The most dramatic changewas observed with the Q56P mutant. Pressures below the MscSthreshold routinely elicited openings of Q56P channels (Fig.

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FIG. 1. The relative threshold sensitivities of wild-type and Q56Pmutant MscL. (A) Patch-clamp recordings of channel activities at -20mV from spheroplasts expressing the wild-type (Upper) or Q56Pmutant (Lower) MscL. Pressure in the pipette, relative to atmospheric,is shown. All recordings were performed at -20 mV; for simplicity, wehave shown channel openings as upward even though inward currentswere registered. Note that in the spheroplast expressing the wild type,MscS activity (*) is observed at lower suction than MscL activity (v);for the Q56P mutant, MscL activity (v) is routinely observed at suctionlower than those for MscS activity ((B) A typical experiment whereopen probability (P.) is plotted against pressure stimulation. Pressurewas normalized to the threshold required to open MscS. Both sets ofdata were fit to the equationy = (ex)/(1 + ex), wherex = (p p,i2)/k,p is the applied pressure, p /2 is the point at which the P0 is 0.5, and kis the slope. In this experiment, the p1/2 was 1.3 versus 1.7 and k was7.3 versus 5.4 for Q56P and expressed wild type, respectively. Eachpoint represented =10 sec of recording to avoid over-exercising thepatch. Values for PO were obtained by current integration normalizedto the total number of channels in the patch. At the highest pressures,the current remained relatively constant toward a maximum value, andno opening events were ever observed to exceed the value defined as

P. = 1.0. In the patches from which these data were obtained, thecultures were induced with IPTG for only 10 min (rather than thenormal 2 h) prior to spheroplasting in an attempt to decrease thenumber of channels observed per patch. Eighteen and 60 channelswere observed for Q56P and the expressed wild type, respectively;similar curves, with a consistent change in pi/2 but no consistentchanges ink, were obtained from spheroplasts derived from uninducedcultures containing as few as 3 channels per patch (not shown).

1A Lower). When examined with pressures normalized to theMscS threshold, the open probability of wild-type channels can

be fitted to a Boltzmann distribution as a function of normal-ized pipette pressure. Similar analysis of Q56P channels dem-onstrated a significant and consistent leftward shift of thesensitivity curve, but no consistent change in slope. A typicalexperiment is shown in Fig. 1B.

Several of the amino acid substitutions at positions Q56 andK31 affected channel open times. For example, K31E had

Q56P

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significantly shorter open times, while Q56Y had longer opentimes (Table 1 and Fig. 2). All kinetic data fit well a 3-open-state model with T2 and T3 being the two longer time constants.The shortest T for each group, which was always <0.3 ms, couldnot be measured accurately. Of the mutants showing changesin channel kinetics, the Q56H mutant is especially interesting.This mutant had significantly longer open times at pH 7.5, butnot at pH 5.0 when the histidine was presumably protonated.No such pH-dependent changes were observed with the wild-type channel (Table 1).Western Blot Analysis of Mutants. We compared the ex-

pression levels of wild-type and mutant proteins by isolatingbacterial membranes and performing Western blots on the

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Proc. Natl. Acad. Sci. USA 93 (1996) 11655

solubilized proteins (Fig. 3). All selected point mutants exhib-iting detectable channel activity were found to be expressed atlevels comparable to the expressed wild type regardless of thedegree or nature of the changes in channel activity. Of themutants that had no measurable channel activity, K31I was

observed at the expressed wild type levels, while A2-12 was

expressed at levels lower than the expressed wild type, butgreater than the AW405 parental strain. As expected, A104-136 and A110-136 could not be detected due to deletion of theantigenic region of the protein (not shown). These dataindicate that the differences observed in channel propertiesbetween mutants are not due to different expression levels.

DISCUSSIONWe have generated and analyzed several mutants containingamino acid substitutions or deletions of hydrophilic regions ofthe MscL protein. These engineered mutants were character-ized using a native expression system previously described (7).Although MscL pressure sensitivity can vary appreciably frompatch to patch, presumably due to patch geometry, we foundthat by standardizing recording conditions and using MscS as

an internal control, we were able to obtain consistent values.We have previously proposed a working model for the

structural organization of a MscL subunit (24). Recent struc-tural studies have given credence to the predicted membranetopology, and have suggested that the functional complex is a

homohexamer located in the inner membrane of E. coli (25).In this working model, the first 13 amino acids of the MscLprotein are proposed to form an amphipathic a-helix perhapsimportant in protein folding and/or formation of the pore (Siin Fig. 4). The channel is apparently tolerant of very small(A2-4), but not larger (A2-12) deletions within this amino-terminal domain. In contrast, the MscL protein is quite

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FIG. 2. Single amino acid substitutions in MscL can have drasticeffects on channel kinetics. Open time distributions of the expressedwild type (Top) and mutants K31E (Middle) and Q56Y (Bottom) are

shown. Typical traces from spheroplasts for each strain are shown as

Insets; we have shown the inward current of the channel openings as

upward. The histograms are each fit to a three-open-state modelshown as three component curves beneath the time-distribution curve,the mean and SEM of which are given in Table 1.

FIG. 3. Western-blot analysis for MscL expression in total mem-branes from wild type and several MscL mutant E. coli strains showingcomparable production of MscL proteins. A Western blot usingantibodies against a carboxyl-terminal peptide of MscL is shown.Selected mutants that exhibited altered channel properties (Table 1)were examined. The controls include the MscL null mutant (AMscL),the parental wild type (WT), and the expressed wild type (expressedWT). Plasmid-borne mscL produced more MscL protein than chro-mosomal mscL in wild type due to higher copy number and inductionwith IPTG. Mutants are as labeled in Table 1.

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FIG.4. A working topologic model for a MscL subunit. A schematic of a MscL subunit consistent with the data is shown with its domains labeled.The amino-terminal region (Si) is proposed to form a cytoplasmic amphipathic a-helix. From PhoA fusion studies (25) and the hydropathy plot,we have predicted the transmembrane domains, Ml and M2, to span approximately from L19 to A38 and from A70 to I96. The subunit containsone periplasmic loop, S2-S3, of undetermined structure. The carboxyl-terminal domain (C) resides in the cytoplasm. The positions of mutagenesis,A2-4, A2-12, R104 (for A104-1346), A110 (for A110-136), K31, and Q56, are marked.

tolerant of large carboxyl-terminal deletions; 27 amino acidscan be deleted without major changes in channel function.We have found two residues that reside in regions whose

conformational integrity is required for normal channel gating,Q56 and K31. Our structural data (25) indicate that Q56resides in the periplasmic region. The structure of the proteinin this region, between Ml and M2, remains unclear. ResidueK31, is in a hydrophobic region of the molecule and is thoughtto reside in the middle of Ml. Single residue substitutions ofthese amino acids can lead to changes in MscL channel gating.Sachs (2) has proposed a energetic model for MS-channelgating in which the closed state is separated from the openstate by a single barrier; the difference in energy between openand closed states as well as the height of the barrier depend onmembrane tension. Accordingly, both PO and transition rateconstants are dependent upon tension. Although the prelim-inary kinetic analysis presented here suggests more than oneopen state, this simplified model may help in qualitativelyinterpreting the data. The mutations presented here influencethe equilibrium (P.) and/or kinetic parameters (T) of thissystem. Thus, the mutants with an increased mean open time(Q56 C, F, W, or Y) would have a significantly higher barrier(energy of transition state)-i.e., once in the open state, thesemutants would resist the transition, or intermediate states, thatlead back to the closed state. It is interesting to note that thesubstitution of amino acids containing aromatic rings (F, W,and Y) at Q56 leads to the strongest perturbations in this classof mutants. In contrast, the mutant K31E, which introduces areversal of charge in Ml and yields significantly shorter meanopen times, apparently has a lower transition barrier, makingit much easier for the open channel to achieve a closedconformation. The Q56P mutant has a shifted sensitivitycurve, opening at lower pressure, suggesting a decrease of freeenergy of transition without change in the kinetic barrier; inother words, this mutant appears to have a destabilized closedstate. On the other hand, AQ56 requires significantly morepressure for its gating suggesting an increase energy fortransition from the closed to open state-i.e., the closed stateappears to be stabilized.

Presently, MscL is the only available system to study themolecular basis of MS-channel activity. The work reported

here has narrowed our search for structural domains importantfor MscL-channel activity to 106-amino acid residues, andsuggested that the structural integrity of regions containingQ56 and K31 are critical for normal channel gating.

We are indebted to P. C. Moe, B. Martinac, A. C. Le Dain, andGyorgyi Szebenyi for their helpful comments and critically evaluatingmuch of the manuscript. This work was supported by NationalInstitutes of Health Grant GM47856 to C.K. and National Aeronauticsand Space Administration Grant NAGW-4934 to S.I.S.; P.B. is aDepartment of Energy-Energy Biosciences Research Fellow of theLife Sciences Research Foundation.

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Proc. Natl. Acad. Sci. USA 93 (1996) 11657

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