Proc. Natl. Acad. Sci. USAVol. 90, pp. 11743-11747, December 1993Neurobiology

IgG from amyotrophic lateral sclerosis patients increases currentthrough P-type calcium channels in mammalian cerebellar Purkinjecells and in isolated channel protein in lipid bilayer

(barium current/neurodegeneratlon/cell death)

R. LLINAS*t, M. SUGIMO01*, B. D. CHERKSEY*, R. GLENN SMITHS, 0. DELBONOt, E. STEFANIt§,AND S. APPELT*Department of Physiology and Biophysics, New York University Medical Center, 550 First Avenue, New York, NY 10016; tDepartment of Neurology and§Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030

Contributed by R. Llinds, September 1, 1993

ABSTRACT The effect of the IgG from amyotrophic lat-eral sclerosis (ALS) patients was tested on the voltage-dependent barium currents (IB.) in mammalian dissociatedPurkinje ceils and in isolated P-type calcium channels in lipidbilayers. Whole cell clamp of Purkinje cells demonstrates thatALS IgG increases the amplitude ofIBh without modifying theirvoltage kinetics. This increased IBa could be blocked by apurified nonpeptide toxin from Agelenopsis aperta venom (pu-rified funnel-web spider toxin) or by a synthetic polyamineanalog (synthetic funnel-web spider toxin) and by a peptidetoxin from the same spider venom, ai-Aga-IVA. Similar resultswere obtained on single-channel recordings from purifled Pchannel protein. The addition of ALS IgG increased single-channel In.open time without affecting slope conductance. Theresults described above were not seen with normal human IgGnor with boiled ALS IgG. It is concluded that ALS IgGenhances inward current through P-type calcium channels.Since P-type Ca2+ channels are present in motoneuron axonterminahs, we propose that the enhanced calcium currenttriggered by ALS IgG may contribute to neuronal damage inALS.

Amyotrophic lateral sclerosis (ALS; "Lou Gehrig's dis-ease") has been proposed to be an autoimmune disease thatcan attack upper and lower motoneurons (1), leading toparalysis, respiratory depression, and death. It has beenpreviously reported that IgG from ALS patients has a block-ing effect on L-type calcium channels (2) and in lipid bilayer(3). In addition, ALS IgG enhances calcium currents (Ica) inimmortalized motoneurons (4), acting on an as yet unidenti-fied calcium channel. This IgG has also been shown toproduce an increase in acetylcholine release (5), a mechanismin which the P-type calcium channel is directly involved (6).Here, we demonstrate, using patch-clamp recordings fromPurkinje cells (7, 8), that ALS IgG produces an increase inbarium current (IB.). A parallel experiment using purifiedP-type calcium channels isolated from the cerebellum andincorporated into a lipid bilayer (9, 10) demonstrated thatALS IgG increased the open time of such P-type calciumchannels. These results indicate the presence, in ALS pa-tients, ofan antibody that affects P-type calcium channels byincreasing their open time, without affecting single-channelconductance.

METHODSPurkinje Cells. Purkinje cells were isolated from adult

guinea pig cerebellar cortex and dissociated using the Kay

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and Wong method (11). Following dissociation, cells wereplaced in a recording chamber and visualized with standardlight microscopy. Inward currents were obtained underpatch-clamp conditions in the presence of 1 ,uM tetrodotoxinand of 10 mM Ba replacing the usual 2.0 mM calcium in thebathing solution (12, 13). The patch electrode contained 110mM tetraethylammonium chloride, 4 mM MgCl, 2 mM TrisATP, 1 mM GTP, 14mM creatinine phosphate, 9 mM Hepes,and 9 mM EGTA. Results were obtained using a Wermerpatch-clamp amplifier (PC-501 patch clamp with a I-Gflheadstage; Hamden, CT). The barium current-voltage (I-V)relation was determined prior to the bath administration ofALS IgG or calcium blockers. Following initial measure-ments of IBa in dissociated Purkinje cells (n = 10), IgG wasintroduced directly to the bath at a concentration of 27 pg/ml(patient 1) or 16 Mg/ml (patient 2). In addition to ALS IgGs,IgGs from normal subjects were utilized as controls. Todetermine whether the effects were due to ALS IgG, the IgGwas boiled 20 min. In a second set of experiments IgG waswarmed to 53°C for a period of 30 min to destroy thecomplement, while keeping intact the IgG fraction. P-channelblocker, purified funnel-web spider toxin (pFTX; 1 ,ul/ml)(9), synthetic FTX (sFTX; 1 mM) (10), co-Aga-IVA peptide(50-100 nM) (14), w-conotoxin (10 ,uM), and nifedipine (10,uM) (15) were utilized in the pharmacological studies.Animals/Membranes for P-Channel Isolation. Adult Hart-

ley guinea pigs (200-300 g) from Camm Research Institute(Wayne, NJ) were decapitated with a small animal guillotineunder sodium pentobarbital (Nembutal, 40 mg/kg, i.p.) an-esthesia. The brain was carefully excised and the cerebellumwas dissected free of remaining brain tissue using a smallspatula. The excised cerebellum from 6-12 animals waspooled and homogenized into 10 volumes of 400 mM su-crose/5 mM Tris HCl/0.1% bacitracin/0.1% phenylmethyl-sulfonyl fluoride/5 mM EDTA/-2 units of aprotinin at 4°Cuntil a uniform homogenate was obtained. The homogenatewas then subjected to differential centrifugation at 700 x g for10 min. The supernatant was recovered and subjected tofurther centrifugation at 12,000 x g for 15 min.The supernatant was again subjected to centrifugation at

47,000 x g for 30 min. The pellet was saved and wasresuspended into 100 mM sodium citrate buffer (pH 7.4) to aprotein concentration of =20 mg/ml as determined by Lowryassay. This solution was then brought to 3% sodium choleatebased on the total solution volume. The solution was stirredovernight at 4°C to solubilize protein and was then centri-

Abbreviations: ALS, amyotrophic lateral sclerosis; I, current; V,voltage; FTX, funnel-web spider toxin; pFTX, purified FTX; sFTX,synthetic FTX.tTo whom reprint requests should be addressed.

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fuged at 47,000 x g for 30 min. The supernatant was subjectedto affinity purification as described below.

Afminty Gel Preparation and Protein Purification. An affin-ity gel based on sFTX was synthesized as described (9, 10).The solubilized protein solution was applied to the poly-amine-coupled gel and was stirred overnight at 4°C. The gelmixture was washed with 10 volumes of sodium citrate (100mM, pH 7.4) and was vacuum filtered. To remove the boundprotein the gel cake was resuspended into 100 ml of 1 M CaCl2(pH 7.0) containing 1% sodium choleate and was stirred in acold room for 2 hr. The preparation was vacuum filtered andthe filtrate was collected. The gel was again suspended in theCaCl2/choleate solution and the elution procedure was re-peated. The eluates were pooled and dialyzed for 24 hragainst 100 mM sodium citrate (pH 7.4) to reduce the calciumand detergent concentrations. This solution was then con-centrated to a small volume by dialysis against polyethyleneglycol (Mr 35,000). A portion of this solution was taken forSDS/polyacrylamide gel electrophoresis and the remainderwas reconstituted into lipid vesicles.

Vesicle Formation. Lipid vesicles containing the isolatedP-channel protein were formed using a 4:1 mixture of phos-phatidylethanolamine/phosphatidylcholine in 400 mM su-crose containing 1% sodium choleate by sonication anddialysis as described by Racker (16).

Lipid Bilayer Studies. The functional activity ofthe P-chan-nel protein purified as described above was studied using the"tip-dip" bilayer technique (17). Bilayers were formed ontwo-pull micropipettes with opening diameters of "'1 Jimusing a 1:1 mixture of phosphatidylserine/phosphatidyleth-anolamine. Voltage was applied via the micropipette using anAxon Instruments AI 2120 patch clamp with a 100-Gilheadstage. The bathing solution was held at ground.Data obtained in the channel studies using P-channel

protein preparations of cerebellar origin were amplified to 1V/pA and the membrane current was recorded. To obtainamplification of the Ca2+ channel response, a solution con-

taining 80 mM BaC12 (pH 7.2) was used on the pipette side ofthe membrane and a solution containing 120 mM CsCl (pH7.2) was used on the bath side. The applied voltage (holdingpotential) ranged from -200 mV to +200 mV. In experimentstesting the effects of IgGs obtained from patients with ALS,dilutions of 1:1000 and 1:10,000 were prepared in 80 mMBaCl2 and were used to fill a fresh micropipette. Additionally,experiments were performed using IgGs that had been com-plement-inactivated or totally inactivated by using the heat-ing protocols previously described. Prior to each test of theIgGs, control recordings using pipettes containing only BaC12(pH 7.2) were obtained. Data was filtered at 1000 Hz andanalyzed using the PCLAMP suite of analysis programs (AxonInstruments).

Isolation of IgG from ALS Patients. IgG was purified bypreviously described methods (18) and stored at -80°C untiluse. IgG content in purified fractions was typically >90%o, asmeasured densitometrically from Coomassie-stained SDS/PAGE electrophoretic proffles of IgG fractions and by IgGimmunoprecipitation using a Technicon RA-1000 systemanalyzer (Technicon).

RESULTSPatch-Clamping of Dissociated Purkinje CeHls. Barium cur-

rents obtained from a dissociated Purkinje cell with extra-cellular Ba concentrations of 10 mM are illustrated in Fig. 1A-D. These currents were obtained following various ampli-tude voltage steps from a holding of -70 mV. The currentsdemonstrate the typical slow onset, lack of inactivation, andfast tail current observed in Purkinje cells (7, 8). In Fig. 1Athe central inward current (control) is determined prior to theaddition ofALS IgG. Fifteen minutes after application of theALS IgG, the same voltage step demonstrates an increasedbarium current. Note the significant increase in the amplitudeofthe sustained calcium current as well as in amplitude ofthetail current. A detailed I-V family for IBa before (Fig. 1B) and

A 2-

0 sFTX

-2- control

-4- /i

-6' ALS Ig G(patient 1) |

-8 -___I0 10 20 30

ms

C2 -

0 j ALS Ig G (patient 1)--

B 2

0

nA

1150 mV

-21

-4-

-6]

-8L0

D

nA-2 -

nA-4 l

-6 1:

E

10 20nm

30V

II 1.

2-

O - sFTX

-2-

-4

-6

60 40 0 -40 -60 mV

//

z- ./ Ba++ l0mM

/2; a control, * ALS 19 G

/ a sFTX

-4nA

-_

0 10 20 30Ms

-_0 10 20 30

ms

FIG. 1. Whole cell patch-clamp recording of IBa from guinea pig Purkinje cells in the slice. (A) Lower set of traces, control IBa producedby voltage step of 60 mV from a holding of -70 mV. Following the application of ALS IgG, IBa increased in amplitude. Application of sFTXto the bath abolishes IBa. (B-D) Family of IBa for different voltage steps for control (B), ALS IgG superfusion (C), and after sFTX superfusion(D). (E) I-V relations for the set of records shown in B-D.

T

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after ALS IgG (Fig. 1C) demonstrates that the increase inbarium current is present particularly at levels positive to-40 mV membrane potential. Application of sFTX (Fig. 1 Aand D) demonstrates that this polyamine blocks the inwardBa current. In Fig. 1E, comparison of the I-V relation for thecontrol 'Ba and that obtained following IgG perfusions isshown. Open circles represent the I-V relation for the controllevels and closed circles represent the relation following IgGadministration. These records are typical for the findingsobtained in 10 neurons and indicate that the calcium currentis increased in the presence of IgG.A similar set of recordings obtained for a second patient in

two other Purkinje cells is shown in Fig. 2A. As in the resultsillustrated in Fig. 1, the IBa was blocked by sFTX. In addition,50 and 100 ,uM w-Aga-IVA produced an equally dramaticblock of the Ba where the difference between control ofbarium level and that obtained 12 min after application ofALS IgG is illustrated for another cell.P-Type Channels Are Involved in the Increased Calcium

Entry. To determine the type of voltage-dependent calciumchannel responsible for the increase in IBa, pFTX, sFT7X(known to block P channels) (9, 19), and w-Aga-IVA peptide(14) were tested for IBa-blocking ability. Following applica-tions of ALS IgG, sFTX (at 500 i&M) produced a total blockof the barium current (Figs. 1 A, D, and F and 2 A and B). Inthe record illustrated in Fig. 2B, a set of results similar tothose in Fig. 2 A and C was obtained following w-Aga-IVAsuperfusion. Barium current in this case was blocked byco-Aga toxin IVA at a concentration of 100 nM.To verify that the above results were due to the presence

ofALS IgGs, a similar set ofresults was obtained using boiledIgGs (Fig. 3A), which produced no demonstrable effect onbarium currents. By contrast, ALS IgG heated to 53°C, toinactivate complement, demonstrated a clear increase ofcalcium current, which once again was blocked by pFTX ata concentration of 1 ,u/ml. Bath application of normal IgG insimilar concentration did not produce any change in IBa (Fig.3C). To substantiate further our conclusion regarding

A

0

-2

nA-4

-6

P-channel activation as the possible underlying mechanismfor the increased ALS current, c-conotoxin and nifedipinewere also used at 10 uM and 10 ,uM, respectively. Theseblockers had no effect on IBa, confirming the P-type origin ofthe calcium conductances in Purkinje cells (9, 13).

Lipid Bilayer Results. As previously demonstrated (9, 10),protein isolated from the cerebellar cortex using a sFTX-based affinity gel exhibited single-channel events when ves-icles incorporating the purified protein were studied using thelipid bilayer technique. Fig. 4A shows the recordings ob-tained when vesicles incorporating purified P-channel proteinwere fused with a lipid bilayer in the absence and presence ofIgG obtained from an ALS patient.

In Fig. 4A, normal recordings were obtained at holdingpotentials of -60, -50, -30, and -10 mV in asymmetric 80mM BaCl2/120 mM CsCl solutions. At -60 mV, few if anyopenings were observed. Beginning at -50 mV, channel-likeevents could be detected, which increased in frequency in avoltage-dependent manner as the membrane was furtherdepolarized. Thus, open probability increases with depolar-ization. However, the open times of the individual eventsremain short, with a mean open time of 1 ms (9, 13), and areconsistent with the properties of the P-type calcium channelresponsible for the calcium conductance of mammalian Pur-kinje cells.

Fig. 4B shows the dramatically different recordings thatwere obtained when ALS IgG at a concentration of 3 ,g/mlwas present at the cytoplasmic face of the P-type calciumchannel. The recording obtained at a holding potential of -60mV shows that the threshold for activation ofthe channel hadnot been altered and that little or no activity was apparent. Asthe membrane was depolarized, channel-like events wereobserved. Recordings at -50 mV remain similar to thoseobtained for this channel in the absence of the IgG. Withfurther depolarization, events were seen that are remarkablefor the duration of their open time, which was increasedmanyfold without any alteration of the single-channel con-ductance. Determination of the single-channel current indi-

B

boiled ALS Ig GI(patient 1)

control

nA

0 10 20 30

ms

C

0

nA-2-

-4-

-6

0 10 20 30ms

.1 100 mV

0 10 20ms

30

FIG. 2. Purkinje cell IBa. (A) Effect of boiled ALS IgG compared to the control condition. (B) Effect of IgG heated 53°C and the action ofpFTX on IBa. (C) Effect of normal IgG on IBa. Holding potentials were the same as in Fig. 1.

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A

nA

B

0 10 20 30ms

1- L 1150 mV0-

W-AGA IV

A-1- ,nA-2- control

-4- ALS Ig G (patient 2)

0 10 20 30ms

FIG. 3. Purkinje cell IBa. (A) Control IBa is increased afterexposure to ALS IgG from a second patient. IBa is blocked by sFTX.(B) Similar set of recordings as inA from a different neuron using thesame IgG but blocked by wo-Aga-IVA.

cates that ALS IgG produced no increase in channel con-ductance (Fig. 4C). IgGs from normal subjects and boiled

A2+

Ba

ALS IgG were without effect in these experiments. Theseresults are taken to indicate that ALS IgG acts directly onchannel protein, rather than by other mechanisms involvingintracellular machinery known to regulate single-channel-calcium properties.

DISCUSSIONThe present findings indicate that IgG from ALS patients actsdirectly on P-type channels and modifies the average opentime such that a rather large increase in 'Ba may be observed.This increase does not seem to modify kinetics of the onsetof calcium currents and has little effect on the duration of thetail current. This last point, however, cannot be qualitativelyinterpreted as dissociated neurons retain dendritic branchesthat prevent a proper control ofIBa currents, especially at thebreak ofthe voltage pulse. The results indicate, nevertheless,that ALS IgG as well as complement-inactivated serum arecapable of producing the IBa increase, whereas normal orboiled serum does not modify 'Ba.From the above it may be concluded that IgG affects the

well-known P-type calcium currents in Purkinje cells. Toconfirm this finding, pFTX from Agelenopsis Aperta, sFTX,and w-Aga-IVA toxin were tested and showed calcium-current block in the absence and presence ofALS IgG. Theseresults confirm that pFTX, sFTX (9,13), and w-Aga-IVA (14)can block barium currents in Purkinje cells and demonstratethat the increase of calcium current is brought about byactivation of P-channel types of calcium conductance. Thefact that the increased IBa was insensitive to nifedipine as wellas to c-conotoxin rules out the possibility that these currentscould be due to activation ofan N- or L-type calcium channel(15) and may help to explain the increase in ICa obtained withALS IgG in other preparations such as immortalized mo-toneurons (4).

Finally, results on the lipid bilayer indicate that the effectofALS IgG on P channels is direct, since in the lipid bilayer

B 2+Ba

-10mV _

-3OmV -

-50mV .

-6OmV .I 1pAlooms

-3OmV _r

-5OmV -

-6OmV I lpA250ms

C-60 -30 0 30 60

0.0.

V(mV)

0.-0.5

_J-1.0i(pA)

FIG. 4. Single-channel recording from isolated P-channel protein in the lipid bilayer. (A) Voltage-dependent single-channel recordings. (B)ALS IgG increases open time of single-channel current. Note the change in time base for A and B. (C) I-V relations for normal and ALSIgG-treated single-channel currents.

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preparation only the channels responsible for calcium currentare actually present. Of interest at this level is the fact that inthe presence of ALS IgG the open time for single calciumchannels appears to be voltage-sensitive such that as themembrane is depolarized, the average open time substan-tially increases. This is to be expected as multiple openingsoften coexisted (not illustrated). This may help to explain toa certain extent why this time constant of the tail currentincreased by 100% only in the presence of IgG in dissociatedPurkinje cells, even though very large conductance openevents do not seem to be evident as might be expected. Itmust also be remembered, however, that in the real cell, asopposed to the isolated channel in lipid bilayers, otherchanges may enter into consideration.The present findings are generally in agreement with those

previously reported for the actions of ALS IgG on ionicchannels. In those studies, it was demonstrated that the IgGfraction blocks skeletal L-type calcium channels (2, 3).However, as it is known that ALS IgG produces an increasein acetylcholine release (5) and that in the mammal thisrelease is under the control of P-type channels (6), block ofL-type calcium channels could not explain the pathology seenin ALS.The ability of IgG from ALS patients to act on multiple

types of calcium channels is of interest. In addition to thepresent findings regarding the activation of P-type calciumchannels and previous findings on the block of L-type cal-cium channels, the IgG fraction has also been shown to alterthe behavior of w-conotoxin-sensitive channels in oocytes(E.S. and S.A., unpublished observation). These findings,when taken together, suggest that either ALS patients pro-duce multiple antibodies that act on different calcium channelproteins or, more attractively, that a single antibody is beingproduced that acts on an identical epitope present on multiplechannel types. In the absence of a resolved antibody prep-aration this question cannot be fully addressed. It is also notclear from these results whether the antibodies produced bythese patients are uniquely directed toward calcium channelsor ifthe action is secondary to a different immunologic target.The results, however, are quite unambiguous in demon-

strating that ALS IgG acts directly on P-type channels,allowing increased calcium to enter cells, and may go a longway toward explaining cell degeneration in this syndrome. Inparticular, the possibility remains that autoimmune eventsmay trigger a similar type of sustained calcium inflow in otherneurons since the P channel is present throughout the centralnervous system (20). Such calcium-channel misregulationsmay be at the base of many degenerative diseases of thecentral nervous system, as excessive intracellular calcium

concentrations are well-known to result in cell death, as is thecase in Purkinje cells (21), although the presence of highlevels of calcium buffer protein may actually protect suchcells from early damage.

This work was supported by the National Institute on Aging (GrantAG09480 to R.L., M.S., B.D.C.), Cephalon Inc. (S.A.), and TheMuscular Dystrophy Association (to E.S. and S.A.).

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