572

KIDNEY

[33]

[33] P u r i f i c a t i o n a n d R e c o n s t i t u t i o n o f E p i t h e l i a l Chloride Channels B y D O N A L D W . LANDRY, M Y L E S A . AKABAS, CHRISTOPHER R E D H E A D ,

and QAIS AL-AWQATI Chloride channels are found in most cells, in which they exist in the plasma membrane as well as in intracellular membranes. Eleetrophysiological studies of epithelia have shown that these channels are located in the apical plasma membrane in some cells and in the basolateral membrane of others.~ Because the intracellular activity of CI- is above electrochemical equilibrium in epithelial cells and in smooth muscle, CI- channels play a dominant role in the control of the membrane potential and the transepithelial transport of ions. In intracellular membranes such as the Golgi, endosomes, and lysosomes, these channels are present in parallel to the H+-ATPase, an electrogenic proton pump that serves to acidify the contents of the vesicles. The CI- channel plays an important role in regulation of the degree of acidity of these organelles by shunting the membrane potential generated by the ATPase and allowing maximal acidification.2 Chloride channels are also regulated by a variety of second messengers such as protein kinase A, protein kinase C, and intracellular Ca2+.3 Several important diseases are associated with abnormalities of this channel. In myotonia, there appears to be a defect in the muscle C1- conductance. In cholera and secretory diarrhea, cyclic AMP opens the epithelial CI- channel, leading to massive diarrhea. In cystic fibrosis, neither protein kinase A or C can open this channel. With the development of the patch-clamp method it became apparent that C1- channels exhibit a wide variety of eleetrophysiological behaviors. Single-channel conductances vary from 1 pS to several hundred picoSiemens; some channels show marked rectification while others have linear current-voltage relationships, and most channels do not show marked halide selectivity. The following questions then arise: What is the molecular basis of this diversity of electrophysiological behaviors? Are all C1channels derived from a single protein or are there multiple proteins? Are E. Sehlatter and R. Cneger, Pfluegers Arch. 405, 367 (1985); R. L. Shoemaker et al., Biochim. Biophys: Acta 858, 235 (1986); M. J. Welsh and C. M. Liedtke, Nature (London) 322, 467 (1986). 2 j. Gliekman et al., 2", CellBiol. 97, 1303 (1983). 3 R. A. Schoumacher et aL, Nature (London) 330, 752 (1987); M. Li et al., Science 244, 1353 (1989); T.-C. Hwang et al., Science244, 1351 (1989).

METHODSIN ENZYMOLOGY,VOL. 191

Copy~t © 1990by AcademicPress,Inc. Allt~ts of reproductionin any formreserved.

[33]

EPITHELIAL CHLORIDE CHANNELS

573

all proteins derived from a single gene or from a related family of genes? Finally, what is the molecular basis of these diseases? To answer such questions requires purification of the protein and development of reagents, such as antibodies and oligonucleotide probes. The initial stages of development of these reagents are described below.

Strategy for Purification of the Chloride Channel To purify a protein requires a functional assay and for a channel the obvious function is ion transport. However, membrane proteins must be solubilized for purification and to assay from this condition requires either reconstitution or the development of a ligand through which to construct a binding assay. A high-affinity ligand would also allow the use of affinity chromotography. Regardless of whether one uses classical or affinity chromatography, reconstitution of the pudtied proteins and demonstration of the transport function remains the sine qua non of channel purification. Because it is expected that during purification large losses will be incurred, one must also search for starting materials that are highly enriched in C1channel activity and also readily available in large mounts. This usually restricts one to the use of solid organs such as liver, kidney, and brain. Unlike the case of other channels that have been purified, such as the nicotinic acetylcholine receptor, the voltage-gated Na + and Ca2+ channels, and the ),-aminobutyric acid (GABA) receptor, no useful ligands were available for the epithelial Cl- channel. Using compounds that were known to inhibit C1- transport, e.g., ethacrynic acid, bumetanide, anthranilic acids, and stilbene disulfonates, we started by screening these compounds and their derivatives using an assay of voltage-sensitive 3t~21- uptake into bovine renal cortex microsomes.4 Approximately 100 compounds were screened and 15 examined in more detail. The indanyloxyacetic acids (IAA) constituted an active class of compounds and the most potent of these, IAA-94, inhibited C1- transport with a/~. of 1 #M. [3H]IAA-94 was initially generated by acid-catalyzed isotope exchange to a specific activity of 0.6 Ci/gmol. An equilibrium binding assay was developed in which the bound was separated from the free ligand by filtration on glass fiber filters after 18 hr of incubation. The ligand bound to the kidney microsomes with a Kd of 0.6 gM and for a panel of inhibitors the rank order of potency for ~Cl- transport inhibition correlated with [aH]IAA-94 displacement, all suggesting that IAA-94 binds to the channel. These results were described in detail elsewhere.4 4 D. W. Landry, et al., J. Gen. Physiol. 90, 779 (1987).

574

KIDNEY

[33]

We describe our methods for (1) preparation of bovine kidney membranes, (2) an improved radiolabeling of IAA-94, (3) solubilization of the C1- channel while maintaining the ligand-binding function, (4) synthesis of IAA affinity columns, (5) affinity purification of C1- channels from kidney cortex microsomes, and (6) reconstitution of Cl- channels into proteoliposomes. Preparation of Kidney Membranes 4 Adult cow kidney, freshly harvested and cooled on ice, is dissected for superficial cortex slices; usually 85 g is used as starting material. The slices are minced, mixed with 450 ml iced homogenization buffer (250 mM sucrose, 5 mMTris-HC1, 1 mMdithiothreitol, 1 mMEGTA, pH 8.3), and homogenized with a precooled Waring blender set to high speed for 30 sec, off for 15 sec, then again on for 30 sec. All buffers are kept at 4 °. The homogenate is spun at 6000 g for 15 rain, and the supernatant is spun at 32,000 g for I hr. The resulting pellet consists of a brown lower layer and a fluffy white upper layer which is separated with gentle swirling for 5 sec. The fluffy pellet (40 ml) is diluted with 40 ml of buffer A (250 mM sucrose/ 10 mM imidazole/pH 7) and spun at 40,000 g for 1 hr. The pellet is resuspended in 80 ml of 1 M potassiumthiocyanate (KSCN) and after 30 min spun at 40,000 g for I hr. The pellet is resuspended in 20 ml of buffer A containing seven protease inhibitors [50/zg/ml antipain, 2/zM leupeptin, 10 ~ pepstatin A, 100/z31 (p-amidinophenyl)methanesulfonylfluoride, 10 gM iodoacetamide, 100 #M N-tosyl-L-phenylalaminechloromethyl ketone, and 1 mM EDTA] and stored at - 7 0 ° [SH]IAA-94 IAA-94 is tritiated at the Ca to the carboxylic acid group. This proton exchanges, but only under forcing conditions. Thus, IAA-94 in dry tetrahydrofuran be silylated with trimethylsilylchlodde (l.1 Eq) and quantitatively deprotonated with lithium cyclohexylisopropylamide (1.1 Eq) at - 7 8 ° (Fig. 1). The lithium amide is formed in situ from the corresponding amine and n-butyllithium. The silyl ester enolate is quenched with high specific acitivity tritiated water, which also regenerates the carboxylic acid. Acid-base extraction followed by high-pressure liquid chromatography (HPLC) on a Spherisorb 5# octyl column with CH3OH/H20/acetic acid (125:80: 1) elution yields [3H]IAA-94 with specific activity of 12.4 Ci/ mmol. Using acid-catalyzed exchange with [3H]tdfluoroacetic acid at 120 °, a specific activity of only 0.6 Ci/mmol may be obtained.

[33]

EPITHELIAL CHLORIDE CHANNELS

CI

575

0

IAA-94

~ ~'CH~ O-,,,,K~C02H H I "H I I)CI-SIICH3)~

2) LCIA

c~

o

r-~

0,,~. CO~Si(CH3)3 H/r~)Li(~

13HOH

0 ,,~/C023H 3H/"H

C

CL

I

Acid-Bose Extraction 0

~

3H-IAA'94

O~C02H 3HI ~'H FIG. I.Tritiationof indanyloxyaceticacid(IAA)-94.~, pylamidc.

Lithium cyclohexylisopro-

Solubilizationof the Chloride Channel

In preparation for chromatographic purification, a solubilizationsystem was sought that would maintain the capacity of the channel for binding to [3H]IAA-94. In thisbinding assay, aliquotsof vesiclesor solubilized vesicles(150/zl) arc incubated at 4 ° with 0.I/zM [3H]IAA-94. After

576

KIDNEY

[33]

1.5 hr bound ligand is separated from free by rapid gel filtrationthrough a 0.5 × 3 cm G-50 Scphadex column and the eluatc counted for totalbinding in a liquid scintillationcounter (Beckman Instruments, Inc.,FuUerton, CA). Nonspecific binding is defined as that occurring in the presence of I00 # M IAA-94 and the differencebetween totaland nonspecific binding yields specific[3H]IAA-94 binding. A number of detergents were screened by this method, but N-octylglucosid¢ alone was effective.Bovine kidney cortex microsomes at a finalprotein concentration of I0 mg/ml are solubilizcd by dropwisc addition of a freshlyprepared I0% solution of N-octylglucosidc. The optimum solubilizingconcentration is 1.4%. After 90 min at 4 ° the sample is spun at I00,000 ~ analysis of the supcrnatant shows that 60% of the protein and 20% of the binding sites are solubilized. Although a minority of the sitesare solubilized,the degree of solub'dization is stablefrom day to day. Addition of 10% glycerinmaintains the number of solubilizedbinding siteseven afterfreezingat - 70 ° or incubation at 4 ° for 24 hr. The IC~o for ligand displacement by IAA-94 is approximately 2 ~ in the solubilizedvesicles,similarto that in the intactvesicles. Synthesis of an Indanyloxyacetic Affinity Column 5 IAA-92, an indanyloxyacetic acid similar in structure and potency to IAA-94, was chosen as the basis of an affinityligand because of the availabilityof derivatives,IAA-21 and IAA-23, suitablefor coupling (Fig. 2). IAA-21 is reacted through the primary amino group, with eithercyanogen bromide (CNBr)-activated Scpharose or N-hydroxysuccinimide-activated C H Scpharosc (Fig. 3). IAA-23 reacts only with the cyanogen bromide-activated support. Each of the ligand-resin combinations iseffective and further results are from IAA-23 on CNBr-activated Scpharosc. Dry CNBr-activatcd Scpharosc 4B is reswollen and washed on a sinteredglass funnel with 100 vol of ice-cold I m M HCI. After 15 rain the resin is washed with 3 vol of 0.I M NaHCOff0.5 M NaCI/pH 9. IAA-23 dissolved in 2 ml 0. l M NaHCOff0.5 M NaCI/pH 9 is added to the swoUcn resin and the mixture isagitated.At a ratioof 1.5 gmol ligand to 1.0 ml swollen resin, coupling efficiencyis 97%, as demonstrated by depletion of the ligand from the supernatant in serial UV spectrograms (IAA-23 U V ~ = 268 nm). After 48 hr the resin is washed with alternating 0.1 M Tris/ pH 8.5 and 0.1 M sodium acetate/pH 4.5 and stored in 250 m M sucrose/ 10 mM imidazole/pH 7 with 0.02% NaN 3. Before use, the resin is washed with 250 mM sucrose/10 mM imidazole/10% glycerin/0.6% n-octylglucoside/pH 6.0. The IAA-23 affinity resin (1 ml) is incubated with 6 mg of solubilized D. W. Landry, et aL, Science 224, 1469 (1989).

[33]

EPITHELIAL CHLORIDE CHANNELS Cl

577

0

C I ~ R 0

~C%H

CH$

94 R=- - ~ 92 R = ' ~ 25 R = - ~

NH2

21 R : ~

CH2-NH2

FIG. 2. Structures of IAA ligands for affinity chromatography.

bovine kidney cortex microsomes and agitated for 18 hr at 4 ° for 18 hr. The supernatant is depleted of 55% of the [3H]IAA-94-binding sites, but less than 10% of protein, compared to a control incubation without resin. The extent of specific depletion is not improved by increasing the ratio of resin to protein. Decreasing the ligand density to 0.3 #mol/ml swollen resin decreases the specific depletion and increasing the density to 6 #mol/ml resin only increases nonspecific protein binding. Affinity Purification of the Chloride ChanneP IAA-23 resin (1 ml) is incubated with solubilized bovine kidney membrane vesicles (1.5 ml). The mixture is agitated for 18 hr at 4 ° and then transferred to a column (0.5 × 10 cm). The column is washed at a rate of 0.3 ml/min with 35 ml 250 mM sucrose/10 mM imidazole/10% glycerin/ 0.6% n-octylglucoside/100 #M benzoic acid/l% ethanol/pH 6 and then eluted with 7 ml of a solution containing 100/IM IAA-94 in place of 100 p~/benzoic acid, but otherwise identical. Benzoic acid, which does not affect IAA-94 binding, is added to the wash to minimize the perturbation due to 100 pM IAA-94 in the eluant. The seven protease inhibitors added to the vesicles at the time of their preparation are also present during solubilization and affinity chromatography. Extensive washing of the resin before elution is necessary to remove nonspeeitically bound proteins. The last wash fraction and the specific eluate are precipitated by the addition of

KIDNEY

578

[33] NH2

C.I

0 (/~

O.~COz H NM

Sepharosel / ~ O ' ~ N H

IAA-23 )

Sephorose I~ )

CI

OH.CNBr

0

~

~'~

O~CO~H

( ~ "CH~ NH

Sephorose

0 /

.CH2NH2

CI~

C

~ . ~ O-,,,,...jCO2 H

~CH3

,CH2

l

~ "CH3

,.~ / ~ / - ' ~ /

uV

H

C02H 0 II

[AA-21

Sephor°se~"-NH~(CH2)5~C ~ NH 0

/

CH2 CI

0

0

O~C02H FIG.3. SynthesisofIAA-21and IAA-23Sepharoseaffinitycolumns. 3 - 5 vol of acetone and stored at - 2 0 ° for 18 hr. The precipitate is mixed with sample buffer containing 15 mM dithiothreitol and the proteins are separated by SDS gel electrophoresis. A silver-stained gel is shown in Fig. 4, demonstrating the presence of four proteins of M, 97,000, 64,000, 40,000, and 27,000. The yield is difficult to estimate, but from 6 mg of protein

[33]

EPITHELIAL C H L O R I D E

CHANNELS

579

KIDNEY I 4"

.._1

97644027-

FIo. 4. Electrophoresis of purified CI- channel proteins trom kidney. The IAA-23 affinity resin is mixed with solubilized membranes and after 18 hr transferred to a column. The resin is washed and then eluted with a solution containing 100 pM IAA-94. The eluate (+) and last wash fraction (-) are precipitated in acetone and chromatographed on SDS-PAGE. Proteins are detectedby silver stainingof the gel.

loaded on the affinitycolumn, approximately I/~g is elutcd based on the intensityof silverstainingon the SDS gel.

Reconstitution s

The above procedures result in purificationof drug-binding proteins. To demonstrate that one or allof these proteins arc indeed the Cl- channel requires incorporation of the proteins into artificiallipid bilayers and demonstration of CI- channel activity.W e describe below two kinds of rcconstitutionassays that wc have used, one involving vesicleflux and the other a planar lipidbilaycrtechnique. The purifiedproteins are firstincorporated into phospholipid vesiclesby a detergent dialysisprocedure.6 To 6 M. Kasahara and P. C. Hinkle, Proc. Natl. Acad. Sci. U.S.A. 73, 396 (1976).

580

KIDNEY

[33]

form the vesicles, affinity column eluate is concentrated to 1 ml, added to asolectin and N-octylglucoside, vortexed, and placed in a dialysis tube. The addition of other reagents and the composition of the dialysate varies depending on the specific reconstitution assay.

Coreconstitution of Chloride Channels with Bacteriorhodopsin To demonstrate that the purified proteins represented C1- channels we measured voltage-dependent ~C1- uptake into liposomes. Initially, we found that the uptake of the tracer into liposomes was very high and represented a significant problem in terms of signal to noise ratio. It had been previously demonstrated that phospholipid vesicles have a high neutral C1- transport activity. 7 To counteract this problem, we coreconstituted the purified proteins with bacteriorhodopsin, a light-activated electrogenic proton pump. Previous studies had shown that bacteriorhodopsin reconstitutes asymmetrically and that bacteriorhodopsin vesicles, upon exposure to light, generate a membrane potential and a pH gradient oriented positive outside and acid inside, respectively,s Media with high buffering capacity minimize the pH component of the electrochemical gradient, and the membrane potential component can be used to drive 36C1- uptake. The advantage of this coreconstitution is that one is able to start the reaction instantaneously by switching on the light, which should generate a membrane potential immediately. We use this assay to reconstitute crude solubilized vesicles as well as the affinity-purified proteins. Thus, to 0.8 ml of concentrated affinity-purified protein, 0.2 ml of 1 mg/ml bacteriorhodopsin and 10 nag asolectin are added, all in a final concentration of 1.5% n-octylglucoside. The mixture is dialyzed against 100 m M KC1/10 m M Tris/pH 8.0 (2 X 1 liter) for I day and for a further day against 50 raM piperazine-N,N'-bis-2-ethanesulfonic acid (PIPES) titrated to pH 7.0 with Tris base. Following dialysis, the proteoliposomes are frozen to - 80" until used. Immediately before use the vesicles are thawed to room temperature and sonicated for 25 sec in a Branson 2200 bath sonicator. The same method is used to reconstitute unpurified C1- channel proteins with 0.8 ml of kidney vesicles containing 6 mg protein/ml. For measurement of tracer uptake, 2.3/tCi/ml of 36C1- is added to thawed and sonicated liposomes in a glass fluorimetric cuvette. The vesicles are exposed to light from a Kodak 650H slide projector. At desired times, samples (80-120/tl) are removed and eluted through 8-cm glucoY. Toyoshima and T. E. Thompson, Biochemistry 14, 1525 (1975). s D. Oesterhelt and W. Stoeekenius, Proc. Natl. Acad. Sci. U.S.A. 70, 2853 (1973).

[33]

EPITHELIAL CHLORIDE CHANNELS

581

nate-loaded anion-exchange columns (IRN-78, Rohm and Haas Co., Philadelphia, PA) with 1 ml of 250 mM sucrose/10 mM imidazole/pH 7.0 to remove extravesicular ~sCl-. The uptake in the dark is measured from identical aliquots of each preparation and should be linear with time. This uptake is subtracted from the light-dependent flux. Reconstitution of the purified proteins into bacteriorhodopsin vesicles demonstrates a light-induced ~21- uptake (Fig. 5, closed squares). This uptake is increased when a higher amount of purified protein was reconstituted (Fig. 5, closed squares). Both of these uptakes are substantially reduced by pretreatment of the vesicles with valinomycin to collapse any membrane potential generated. The uptake in the presence of valinomycin (Fig. 5B, open symbols) is equal to the uptake in liposomes reconstituted with bacteriorhodopsin, but no purified CI- channels (not shown). Comparison of the magnitude of the ~21- uptake of the crude and purified vesicles suggests that we have purified the proteins by at least a factor of a 1000, assuming that there is about 1/zg of reconstituted purified proteins.

Reconstitution of Chloride Channel Proteins into Planar Bilayers The concentrated purified channel proteins are added to 10 mg of asolectin and 9 mg of N-octylglucoside, vortexed, placed in a dialysis tube (Spectra-por, M r 14,000 cutoff), and dialyzed against 1 liter of 10 mM KC1/700 mM sucrose/10 m M HEPES titrated to pH 7.0 with KOH for 15 hr. The dialysate is changed and dialysis continued for an additional 3 hr. Vesicles are stored on ice until use. 400

his

350 300 E

3eC~-l

/

250

?oI

:if -5

i

0

5

10

15

20

25

T~me, min

FIG. 5. Reconstitution of Cl- channels with bacteriorhodopsin. Less than 1 pg of puritied protein (open or closed circles) or a 10-fold higher amount (open or closed squares) is reconstituted. Valinomycin is added before the fight was turned on (open symbols).

H~

582

KIDNEY

[33] pA 8

.

.

.

.

C

t 1oo

20 MSEC

-8

Fro. 6. Singe-channel recording (left) and I-V relations (fight) of purified C1- channels reconstituted into planar lipid bilayers. The trace shows the channel in 350 mM KCI, in which the single-channel conductance is 70 pS; 26 pS in symmetrical 150 mM KCI. A downward deflectionrepresents channel opening. C represents closed state. There are either three identical channels in the record (or one channel with two subconductance states). Holding potential (in millivolts) is indicated to the fight of the trace. I- V relation for this channel was obtained in the presence of a 150 to 10 mM KCI gradient (A) or with symmetrical 150 mM KCI (X).

Planar bilayers (4% asolectin in decane) are formed by the brush technique in a 100-gin hole9 in a Teflon partition. Vesicles are squirted at the membrane through a micropipet positioned 2 0 - 5 0 g m from the planar bilayer. 1° The cis (vesicle-containing) chamber has a buffer of either 350 m M KCI/10 m M CaC12/10 m M HEPES/pH 7.0 with K O H or 150 m M KC1/400 m M urea/20 m M hemicalcium gluconate/10 m M HEPES/pH 7.0 with KOH. The trans side contains a similar buffer with either 100 r a M KC1 or 10 m M KC1 but without urea. The single-channel currents are amplified with a home-made current-to-voltage converter and amplifier and recorded on a PCM-video tape recorder (Indec Systems, Inc., Sunnyvale, CA). The data are digitized and analyzed using interactive programs on a laboratory computer system (Indec Systems). Records are filtered at 300 Hz prior to digitization through an 8-pole Bessel filter (Frequency Devices). Potentials given are those in the cis chamber relative to virtual ground in the trans chamber. We found only anion channels and one of them is shown in Fig. 6, where one vesicle appears to have incorporated three identical channels with a linear I - V relationship and a single-channel conductance of 30 pS. We cannot exclude the possibility that this tracing represents a single 90-pS channel with two subconductance states of 30 and 60 pS. Other channels with different electrophysiological characteristics were also seen. A. Finkelstein, this series, VoL 32, p. 387. 98, 1063(1984).

io M. H. Akabas et al., J. Cell Biol.

mv

[34]

RECONSTITUTION OF RENAL TRANSPORT PROTEINS

583

Concluding Comments We have applied the above methods to successfully purify and reconstitute C1- channels from a variety of sources, including apical membranes from bovine tracheal mucosa, sarcolemma from rabbit striated muscle, and bovine thyroid. We observed electrophysiologically distinct channels from each source. That apparently different C1- channels from these diverse sources would bind to an IAA affinity column was unexpected. Since a positively charged analog to the IAAs also inhibits C1- transport, IAA compounds do not merely compete for a C1--binding site. This suggests a fundamental structural similarity among the various Cl- channels purified by IAA affinity chromatography. We have scaled our procedures, obtaining adequate quantities of purified proteins for N-terminal sequencing and the generation of antibodies. By means of these reagents, we are investigating C1- channel structure.

[34] R e c o n s t i t u t i o n a n d F r a c t i o n a t i o n o f R e n a l B r u s h Border Transport Proteins B y HERMANN KOEPSELL a n d STEFAN SEIBICKE

Introduction It has been known for many years that several Na+-coupled transport systems exist in brush border membranes of small intestine and renal proximal tubules, which are responsible for the reabsorption of I)-glucose, amino acids, and anions. 1-3 The Na+-~glucose cotransporter is the best investigated Na +-coupled transport system from renal and intestinal brush border membranes, and reconstitution and purification experiments have been nearly exclusively performed with this transporter. 4-17 Since the S. Silbernagl, E. C. Foulkes, and P. Dec~jen, Rev. Physiol. Biochem. Pharmacol. 74, 105 (1975). 2 K. J. Ullrich, Annu. Rev. Physiol. 41, 181 (1979). 3 H. Murer and G. Burekhardt, Rev. Physiol. Biochem. Pharmacol. 96, 1 (1983). 4 R. K. Crane, P. Malathi, and H. Preiser, Biochem. Biophys. Res. Commun. 71, 1010 (1976). 5 R. K. Crane, P. Malathi, and H. Preiser, FEBS Lett. 67, 214 (1976). 6 j. T. Lin, M. E. M. Da Cruz, S. Riedel, and R. Kinne, Biochira. Biophys. Acta 640, 43 (1981). 7 W. B. Im, K. Y. Ling, and R. G. Faust, J. Membr. Biol. 65, 131 (1982).

METHODS IN ENZYMOLOGY, VOL. 191

Copyfisht © 1990 by Academic ~ Inc. An dghtsof~produ~ioninany form reserved.

Purification and reconstitution of epithelial chloride channels.

572 KIDNEY [33] [33] P u r i f i c a t i o n a n d R e c o n s t i t u t i o n o f E p i t h e l i a l Chloride Channels B y D O N A L D W . LANDRY...
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