Proc. Natl. Acad. Sd. USAVol. 74, No. 7,3090-3094, July 1977Physiological Sciences

Modulation of acetylcholine receptor by antibody against thereceptor

(muscle/acetylcholine sensitivity/acetylcholine noise/myasthenia gravis)

S. HEINEMANN, S. BEVAN, R. KULLBERG, J. LINDSTROM, AND J. RICE*The Salk Institute, San Diego, California 92112; and * Department of Mathematics, University of California, San Diego, La Jolla, California 92137

Communicated by Stephen W. Kuffler, March 11, 1977

ABSTRACT Antibody against acetylcholine receptor in-duces an increase in the rate of degradation of acetylcholinereceptors on a mouse cell line (BC3H-1) and cultured rat skeletalmuscle. The increased rate of degradation results in a lowereddensity of acetylcholine receptors on muscle membrane and alowered sensitivity to iontophoretically applied acetylcholine.The modulation of acetylcholine receptor is energy, tempera-ture, and time dependent and may be related to antigenicmodulation found in other systems. Acetylcholine noise analysisdemonstrates that antibody against acetylcholine receptor re-duces the channel mean conductance and mean open timeslightly.

It is concluded that antibody binds to the acetylcholine re-ceptor, impairs its function, and induces receptor degradation.This results in a lowered density of acetylcholine receptor anda lowered sensitivity to acetylcholine. Patients with myastheniagravis have antibodies to their acetylcholine receptor in theirserum. Antigenic modulation of receptor in the muscle of pa-tients with myasthenia gravis could contribute to the observeddecrease in amplitudes of miniature endplate potentials andin muscle acetylcholine sensitivity, and the symptoms of mus-cular weakness.

The regulation of the density and distribution of the acetyl-choline (ACh) receptors found on skeletal muscle has been ofgreat interest to biologists because it may reflect a fundamentalprocess important in synaptogenesis (1, 2). In this paper wedescribe a form of regulation that occurs when muscle is ex-posed to antibodies directed against purified ACh receptor. Ourexperiments show that antibody against receptor induces anincrease in the rate of degradation of ACh receptors and alowered sensitivity to iontophoretically applied ACh. Ourprevious work has shown that skeletal muscle from rats im-munized against purified ACh receptor (torpedo or electric eel)has a reduced sensitivity to ACh (3), exhibits reduced miniatureendplate potential amplitudes (3, 4), and contains decreasedamounts of receptor (5). In immunized rats, antibody againstrat receptor is found both in the serum (6) and bound to muscleACh receptors (5). Serum taken from such immunized rats andapplied to muscle in vitro reduces the sensitivity of the muscleto ACh (3).Human patients with myasthenia gravis also have antibodies

against the ACh receptor in their sera (6-10) and bound to theirmuscle ACh receptors (J. Lindstrom and E. Lambert, unpub-lished data). Furthermore, their muscles exhibit reducedminiature endplate potential amplitudes and a reduced sensi-tivity to ACh (11-13) and contain decreased amounts of re-ceptor (ref 14; Lindstrom and Lambert, unpublished data).The following experiments were performed to determine the

mechanism by which antibody against the ACh receptor re-duces muscle sensitivity to ACh.

MATERIALS AND METHODSCell Culture. The clonal mouse muscle cell line BC3H-1 (15,

16) was grown in Dulbecco's modified Eagle's medium con-taining 10% fetal calf serum (FCS). The cells were allowed toreach the stationary phase and were used for experimentswithin 10 days.

Rat primary skeletal muscle cultures were prepared from 17-to 20-day Lewis rat embryos. Limb muscle was dissected fromskin, bone, and connective tissue and minced into small pieces(1 mm3). The tissue was dissociated enzymatically as describedelsewhere (17) except that the Viokase treatment was omitted.Cells from 12 limbs were plated on 40-60 35-mm tissue culturedishes in Dulbecco's modified Eagle's medium containing 10%FCS. The myoblasts fuse, forming myotubes within 3-4 days,and were used for experiments within 10 days.

Preparation of Antiserum against ACh Receptor. Lewisfemale rats were injected with 100 pmol of ACh receptor pu-rified from Electrophorus electricus. Freund's complete ad-juvant plus Bacillus pertussis were injected as adjuvants. After4 weeks these rats enter a chronic phase of weakness and havehigh titers of antibody in their sera (6). The sera from 160 im-munized animals were pooled and used as antiserum againstACh receptor. The pooled serum was titered for antibodyagainst rat ACh receptor by immunoprecipitation as described(6). It had a titer of 1.1 x 10-7 M against rat ACh receptor.Adjuvant serum was prepared from rats injected only withadjuvant and had no detectable (<10-9 M) antibody againstrat ACh receptor. Rat gamma globulin was prepared by pre-cipitation of rat serum with 35% (NH4)2SO4. The precipitatewas dissolved in phosphate-buffered saline and chromato-graphed on a 1.5 X 111 cm Ultrogel ACA44 column. Thegamma globulin peak was concentrated by an Amicon de-vice.

Electrophysiology. Muscle sensitivity to ACh was measuredby standard iontophoretic techniques (2). The culturedmyotubes were penetrated with an intracellular recordingelectrode and the voltage response to iontophoretically appliedACh was measured. All recording was done at 21-23o in saline(142.6 mM NaCl/5.6 mM KCI/10 mM CaCl2/5 mM N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid-NaOH, pH7.4). The iontophoretic electrode was filled with 3 M AChC1and had a resistance of 150-250 MU. The iontophoretic elec-trode was placed on the surface of the muscle at a random po-sition. The backing current (1 to 3 X 10-9 amp) on the AChelectrode was adjusted to give the maximum response. Onepulse size was used for each fiber (usually 10-8 amp; 0.2-1.0ms). Dose response curves were not obtained. See Table 2 foifurther details.

Preparation of 125I-Labeled a-Bungarotoxin ("251-a-BuTx).a-BuTx was purified from the venom of the krait Bungarusmulticinctus, Ross Allen Serpentarium. The a-BuTx was io-dinated by the iodine monochloride method (18). The diiodo-

3090

Abbreviations: 125I-a-BuTx, 125I-labeled a-bungarotoxin; Dnp, 2,4-dinitrophenol; FCS, fetal calf serum; ACh, acetylcholine; prep medium,Dulbecco's modified Eagle's medium in which the bicarbonate is re-placed with 1.08 mM Na2PO4/1.5 mM KH2PO4.

Proc. Natl. Acad. Sci. USA 74 (1977) 3091

Table 1. Loss of 1251-a-BuTx binding to muscle cells (BC3H-1)treated with antiserum against ACh receptor

RelativeIncubation 125I-a-BuTx

Serum conditions binding I SEM

Control 370 1.00Adjuvant 370 0.92 ± 0.09 (13)Anti-ACh receptor 370 0.29 + 0.08(10)Anti-ACh receptor 370, Dnp/NaF 0.86 ± 0.06 (2)Anti-ACh receptor 21-230 0.82 I 0.06 (3)

BC3H-1 cells were incubated for 2-3 hr in Dulbecco's modifiedEagle's medium, in which bicarbonate was replaced with 1.08 mMNa2PO4/1.5 mM KH2PO4, (prep medium) plus serum. Control, 12%FCS; adjuvant, 2% rat adjuvant serum plus 10% FCS; anti-ACh re-ceptor, 2% antiserum against ACh receptor plus 10% FCS. In somecases the rat serum was heated to 560 for 30 min to inactivate com-plement. After the incubation in serum the cells were washed in prepmedium plus 0.2% FCS and incubated in 10-8 M 125I-a-BuTx for90-120 min at 21-230. The cells were scraped off the dish, collectedon a Celotate filter (Millipore), and washed twice with prep mediumplus 0.2% FCS and five times with phosphate-buffered saline. Theradioactivity of the filter containing the cells was determined in aliquid scintillation counter. In all cases 1251-a-BuTx binding was in-hibited more than 85% by 1 mM d-tubocurarine. Each experimentwas done with duplicate or triplicate samples; numbers in parenthesesare the number of times the experiment was performed. All values arenormalized to the 1251-a-BuTx binding to control dishes determinedin each experiment with sister cultures. Energy metabolism wasblocked by adding 5mM Dnp plus 2mM NaF 3.5 hr before serum wasadded. Cell counts demonstrated that the serum treatment did notsignificantly alter the number of BC3H-1 cells remaining on the dish(±10%).

a-BuTx was purified by chromatography (16) and used in theseexperiments. The ability of muscle cells to bind 125I-a-BuTxwas measured by an assay described elsewhere (19).

RESULTSA model system utilizing muscles grown in tissue culture wasused to study the effect of antibodies against receptor on AChreceptor density and function. Two sources of muscle were used(i) a clonal cell line BC3H-1, which has a high density of nico-tinic ACh receptors in its membrane (15, 16), and (ii) primaryskeletal muscle cultures from rat (Lewis) limbs. Antisera againstACh receptor were obtained from rats (Lewis) immunized withpurified eel ACh receptor (4-6).The ACh receptor density and degradation rate were de-

termined by measuring the muscle membrane's ability to bindand then degrade bound 125I-a-BuTx. a-BuTx is a protein(molecular weight 8000) isolated from the venom of the kraitthat binds specifically and with high affinity to the ACh re-ceptor (20). The degradation of bound 125I-a-BuTx is an ac-curate measure of the turnover of ACh receptor (21, 22). Re-ceptor function was assayed by the iontophoretic applicationof ACh to the muscle surface, and the properties of individualACh receptor channels were determined by means of ACh noiseanalysis (23, 24).

Effect of Antisera against ACh Receptor on the Numberof a-BuTx Binding Sites. The ability of both BC3H-1 andprimary rat muscle cells to bind 1251-a-BuTx is reduced by in-cubation at 370 for 2-3 hr in medium containing antiserumagainst ACh receptor (Table 1). Serum from rats immunizedonly with adjuvant had no effect when compared to controlsthat were incubated in fetal calf serum (Table 1).The antibody-induced loss of a-BuTx binding capacity is

blocked by a mixture of 2,4-dinitrophenol (Dnp) plus NaF and

0-10.2-0.3--

0.4-

0 5-

.,- 0.6-

- 0.7-

0.8-

A

."I~~~~~~~~

02-

5 0.4-3 I.

0 50 100 15 200 250 300

Time (minutes)FIG. 1. Kinetics of 125I-a-BuTx loss from BC3H-1 cells. (A)

BC3H-1 cells were incubated in 10-8M 125I-a-BuTx for 2 hr at 21-23°in prep medium containing 0.2% FCS. The cells were washed fourtimes in prep medium containing 0.2% FCS and then put into prepmedium containing various sera and incubated at 370. (B) Parallelexperiment to A except the cells were poisoned with 5mM Dnp plus2 mM NaF. The Dnp/NaF was added 1 hr before 125I-Ca-BuTx wasadded and was present throughout the experiment. (0) Control, 12%FCS; (A) 2% rat adjuvant serum plus 10% FCS; (0) 2% rat antiserumagainst ACh receptor plus 10% FCS. In the presence of 1 mM d-tu-bocurarine the 1251-a-BuTx binding was inhibited by more than 82%.Each point was determined by averaging the results from threeidentical cultures. The variation between sister cultures was alwaysless than ±15%.

is much reduced at 21-23° (Table 1). These experiments showthat the loss of a-BuTx binding capacity is not the result of thesimple binding of antibody to the receptor. It must reflect someprocess that requires energy.A direct immunoprecipitation assay was used to determine

the rate of binding of antibody against ACh receptor to surfaceACh receptor. After a 30-min incubation, about 70% of thesurface ACh receptors had antibody bound to them. After 60min, about 86% of the surface ACh receptors had antibodybound to them. The rate of antibody binding was not signifi-cantly effected by temperature (21-23° or 37°).

Loss of 1251-a-BuTx from Muscle Cells Treated with An-tiserum against ACh. Receptor. The reduced binding of a-BuTx after incubation with antiserum against ACh receptor(Table 1) can be explained by an increased rate of degradationof membrane ACh receptor. This was demonstrated by mea-suring the rate of loss of bound toxin in the presence of antise-rum against ACh receptor.

125I-a-BuTx was bound to the muscle (BC3H-1) cells in cul-ture and excess a-BuTx was washed away. The rate of 125I re-lease into the medium from the muscle was measured in thepresence of antiserum against ACh receptor. This experimentdemonstrated that antiserum against ACh receptor stimulatesthe release of 125I into the medium (Fig. 1A). Under controlconditions (12% FCS or serum from rats immunized with ad-juvant) the 125I was released at a rate of 12% per hr (Fig. 1A).This agrees well with values previously reported for the rate of

Physiological Sciences: Heinemann et al.

S802 Physiological Sciences: Heinemann et al.

12SI-.i-BuTx [ 14C ] tyrosine

r0006000

L5000H 4000

13000

2000

1000A

500

400

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100

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Proc. Natl. Acad. Sci. USA 74 (1977)

l25I-a-BuTx release from rat diaphragm muscle in organ cul-ture (25) and the BC3H-1 cell line (16). In the presence ofantibody against the ACh receptor, the rate of 125I release isincreased to 70% per hr (Fig. WA). Similar results were obtainedwhen purified gamma globulin was used, demonstrating thatcomplement is not involved in this reaction (Fig. 2 B and C).

In the presence of blockers of energy metabolism (5 mM Dnpplus 2 mM NaF) there is no stimulation of 125I release by anti-serum against ACh receptor (Fig. 1B). The antibody-stimulatedrelease of 125I cannot therefore be due to the simple dissociationof the 125I-a-BuTx-receptor complex. This is comfirmed by thefinding that most of the 125I in the culture medium is no longerassociated with intact a-BuTx. Media from 125I-a-BuTx-labeledmuscle cultures that had been incubated in the presence ofpurified antibody were fractionated on BioGel P2 columns (Fig.2). This experiment demonstrates that most of the 125I releasedinto the medium is recovered in the included volume of the P2column, the position of low-molecular-weight material (Fig.2 B and C). Furthermore, the increased release of 125I seen inthe presence of antibody against receptor (Fig. 1A) is due to anincrease in the release of small-molecular-weight 125I-labeledmaterial, presumably 125I-labeled tyrosine (Fig. 2C). There isno increase in the release of intact 125I-a-BuTx, as would be thecase if the antibody against ACh receptor simply increased therate of dissociation of the a-BuTx-ACh receptor complex.When 125I-a-BuTx is incubated with antiserum against AChreceptor and with muscle cells in the presence of an antagonistof the ACh receptor (10-3 M d-tubocurarine) no degradationof 125I-a-BuTx is observed (Fig. 2A). This demonstrates thatthere is no substance in antiserum against ACh receptor, ormaterial produced and secreted by muscle, that degrades125I-a-BuTx. Thus, 125I-a-BuTx must be bound to the AChreceptor to be degraded. This finding supports the simplestinterpretation that 1251 release is a measure of the degradationof the 125I-a-BuTx-receptor complex (25, 26). This is confirmedby recent experiments showing that the rate of ACh receptorturnover inferred from 125I release is the same as that obtainedby direct measurement of ACh receptor turnover (21, 22).The slow release of 125I from cells poisoned by Dnp/NaF is

probably due to the simple dissociation of the 125I-a-BuTx-AChreceptor complex since the released 1251 runs in the void volume,the position of native 125I-a-BuTx (Fig. 2D).

At 23' the rate of 125I-a-BuTx degradation is much reduced,and there is no stimulation of degradation by antiserum againstACh receptor (Fig. 2 E and F).

Effect of Antiserum against ACh Receptor on SkeletalMuscle Sensitivity to ACh. Incubation of skeletal muscle(Lewis primary cultures) in the presence of antiserum againstACh receptor at 370 results in a reduction in the number ofca-BuTx binding sites (Table 2). If this reflects a reduction in thedensity of functional ACh receptors and the ACh response isreceptor limited, then muscle sensitivity to ACh should be de-creased by antibody treatment. Table 2 confirms this prediction.More importantly, the ability of antibody to affect muscle AChsensitivity is temperature dependent. At 23-21' little loss of

F

ffi 10 is 0t 25 30Fraoction

35

FIG. 2. Chromatography on BioGel P2 of 125I released from thecultures. (A) Elution profile of 1251-a-BuTx incubated for 7 hr withBC3H-1 cells in the presence of 1 mM d-tubocurarine/10% antiserumagainst ACh receptor in prep medium at 37°. The small amount oflow-molecular-weight material was also seen in the 125I-a-BuTx stock.(B) 125I released from BC3H-1 incubated with control rat gamma

globulin (0.2 mg/ml) for 4 hr in 2% FCS in prep medium at 37°. (C)Parallel culture toB except that the BC3H-1 cells were incubated inrat gamma globulin against ACh receptor (0.2 mg/ml, 7 X 10-9 Mantibody against rat ACh receptor). (D) 1251 released from BC3H-1cells poisoned with 5mM Dnp plus 2 mM NaF. The cells were incu-bated for 3.3 hr in 2% antiserum against ACh receptor and 10% FCSin prep medium at 37°. (E) 125I released from BC3H-1 cells incubatedfor 4 hr at 23° in 2% adjuvant rat serum and 10% FCS in prep medium.(F) Parallel culture toE except that the BC3H-1 cells were incubatedin 2% rat antiserum against ACh receptor and 10% FCS in prep me-dium. In all cases the recovery of 1251 was greater than 85%.

4020

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00 *--so

Physiological Sciences: Heinemann et al.

Table 2. Effect of antibody against ACh receptor on the densityand function of the ACh receptor on cultured rat skeletal muscle

Relative RelativeIncubation 1251-a-BuTx ACh sensitivity

Serum conditions binding ± SEM + SEM

Control 370 1.00 1.00Adjuvant 370 0.98 ± 0.07 (3) 1.03 (1)Anti-ACh

receptor 370 0.50 ± 0.12 (6) 0.42 0.07 (8)Anti-ACh

receptor 370, Dnp/NaF 0.83 0.11 (3)Anti-ACh

receptor 21-230 0.80 ± 0.18 (4) 0.92 0.17 (2)

1251-a-BuTx binding was measured as described in the legend ofTable 1. The time of incubation in antibody varied from 1.5 to 4.5 hr.All experiments were done in 2% FCS plus 10% rat antiserum againstACh receptor (final concentration: 1.1 X 10-8M antibody against ratreceptor). In some experiments purified rat gamma globulin was used(0.2 mg/ml or 7 X 10-9M antibody against rat recpetor) instead of ratserum. The ACh sensitivity was measured as described in Materialsand Methods. Numbers in parentheses are the number of experimentsperformed. In each experiment the ACh sensitivity was determinedon 10 to 20 myotubes. The resting potentials varied from -40 to -65mV. Measured sensitivities were corrected to -60 mV for comparison(reversal potential = 0 mV). The mean sensitivity of control myotubeswas 3034 + 1120 mV/nC (n = 36). The mean input resistance of con-trol myotubes was 32 i 43 MS] (n = 43) and was not significantlychanged by treatment with antiserum against ACh receptor. My-otubes treated with antiserum against ACh receptor had a mean inputresistance of 35 ± 28 MO (n = 58). (n = number of myotubes tested.)This is consistent with previous experiments showing that antibodyagainst ACh receptor has no effect on muscle input resistance or

membrane time constant (3). The myotubes were generally incubatedin serum for 3.5 hr. Similar results were obtained after a 22-hr incu-bation.

muscle sensitivity is seen, while incubation at 370 results in a

58% loss of muscle ACh sensitivity. This agrees well with pre-vious experiments showing a loss of ACh sensitivity of normaldenervated rat diaphragms that had been incubated with an-

tiserum against ACh receptor at 37° (3). Thus, the loss of muscleACh sensitivity and loss of a-BuTx binding seen after antibodytreatment are both temperature dependent and presumablyreflect the same process, degradation of membrane ACh re-

ceptor. Preliminary experiments indicate that the rate of125I-a-BuTx release from primary cultures of skeletal muscleis increased in the presence of antibody.

Effect of Antiserum against ACh Receptor on ACh Re-ceptor Channel Properties. The effects of immune serum onthe conductance and kinetics of ACh receptor channels inskeletal muscle (Lewis primary cultures) were investigated bymethods of noise analysis (23, 24). Recordings of ACh noisewere made from cells treated with adjuvant serum and withantiserum against ACh receptor. Cells were incubated in 10%serum for 1 hr at 370 and recordings were then made at roomtemperature (21-230) in the presence of 10% serum. Underthese conditions 74% of the muscle's surface ACh receptors hadantibody bound to them [determined by Triton extraction anddirect immunoprecipitation with rat immunoglobin preparedin goats (5)].The spectra computed from ACh noise after incubation in

either antiserum against ACh receptor or adjuvant controlserum were well fitted by the equation for S(f) (Table 3). Theestimated values for conductance and kinetics are given inTable 3. There was a slight reduction in both channel conduc-tance and mean open time in cells that were incubated in an-

tiserum against ACh receptor. The mean conductance in cells

Proc. Natl. Acad. Sci. USA 74 (1977) 3093

Table 3. Effect of immune serum on ACh receptorchannel properties

Mean singlechannel Mean open Voltage dependence

conductance* time* of X(pMho ±SD) (msec +SD) (-Aln-r/AmV)

Adjuvantserum 52 + 7 (10) 5.2 + 1.7 (10) 0.0084 + 0.0019 (10)

Immuneserum 44 + 8 (24) 4.0 ± 0.7 (24) 0.0111 i 0.0051 (24)

ACh-induced membrane current fluctuations were recorded fromcultured muscle cells obtained from Lewis rat embryonic skeletaltissue. Cells were rounded up by exposing them to colchicine (10-7M) for 1-2 days before recording. Suitable cells had input resistancesof 5-20 Mg (at 80 mV) and were 50-100 um in diameter. Cells werevoltage clamped using two intracellular electrodes filled with potas-sium acetate (resistances of 5-15 MR). Feedback gains were 900-1200.Clamping currents were measured by a virtual ground current mon-itor. ACh was applied iontophoretically from a micropipette posi-tioned 25-50 /im from the cell. All recordings were done at 230 inmedium containing 150mM NaCl/5 mM KCl/1.8 mM CaCl2/5 mMN-2-hydroxyethylpiperazine-N'-ethanesulfonic acid-NaOH, pH 7.4.Methods of spectral analysis were similar to those reported elsewhere(23). Data were recorded on analogue tape and the Fast FourierTransform was performed on bandpass filtered records (0.3-250 Hz,24 dB/octave rolloff) of 1.96 sec length containing 2048 digitizedsamples. Typically, eight to ten records of ACh-induced noise and fiveto six records of control noise were used to compute each powerspectrum. The spectra were fitted using a least squares method bya function of the form:

S(f) - 2MVT-Y1 + (27rTf)2

where f = frequency, X = mean channel open time, v = channel con-ductance, A = mean current, and V = the clamped membrane po-tential (reversal potential being 0 mV). Numbers in parentheses arethe number of muscle cells examined.*At-60mV.

treated with antibody against ACh receptor was 15% less thanthat of adjuvant-treated cells (P < 0.01, Mann-Whitney non-parametric test), and the mean channel open time was 23% less(P < 0.05). These small differences may represent impededchannel function due to bound antibody. The mean chargepassed through a channel during a single opening in the pres-ence of antibody against receptor was 1.1 X 10-14 + 0.2 X 10-14coulomb. This was about % the mean charge passed by a singlechannel in adjuvant serum, 1.6 X 10-14 0.4 X 10-14 coulomb(P < 0.001).The voltage dependence of channel open time in cells treated

with antiserum against ACh receptor was not significantlydifferent from control values. The ACh reversal potential (0mV) was not changed by antiserum against ACh receptor.

DISCUSSIONThese studies demonstrate that antibodies directed against theACh receptor increase approximately 6-fold the rate of deg-radation of the ACh receptor on cultured BC3H-1 muscle. Thisresults in a lowered density of ACh receptors and a loweredmuscle sensitivity to ACh. The antibody-induced modulationof ACh receptor density is energy, temperature, and time de-pendent. Receptor modulation does not depend upon com-plement since purified antibody stimulates ACh receptordegradation.ACh noise analysis demonstrates that the ACh receptors that

remain functional in the presence of antibody have a smallimpairment in function. Both the channel mean single channel

3094 Physiological Sciences: Heinemann et al.

conductance and mean open time were reduced slightly. Weconclude that the decreased ACh sensitivity is due to two pro-cesses: (i) a small decrease in ACh receptor function due tobinding of antibody and (ii) a decrease in ACh receptor densitydue to an antibody-induced degradation of ACh receptor.

Recently we have analyzed the effect of sera from humanmyasthenia gravis patients on cultured human muscle. Ourresults show that human muscles exposed to sera from myas-thenia gravis patients have reduced muscle sensitivity to ACh.The reduction in sensitivity is temperature dependent and mayinvolve a reduction in receptor density. In addition, sera frommyasthenia gravis patients increase the rate of ACh receptordegradation in the BC&H-1 muscle cell line (Bevan, Kullberg,and Heinemann, unpublished data).We have studied the effect of antisera against ACh receptor

on the ACh receptor found on cultured muscle. Our finding thatthe density of ACh receptors is reduced cannot, at present, beextended to the adult neuromuscular junction. The ACh re-ceptor found on cultured muscle is presumably analogous tothe extrajunctional receptor found on denervated skeletalmuscle. The receptors found at endplates are more stable (25,27) and have several different properties that could affect theirresponse to antibody (28-S0). -With these reservations, however,it is clear that an antibody-induced modulation of ACh receptorleading to a lowered density could contribute to the changesthat occur in muscle in immunized rats as well as in humanmyasthenia gravis patients. Reduced miniature endplate po-tential amplitudes and reduced muscle sensitivity to ACh wouldall be consequences of receptor modulation. Rats immunizedagainst ACh receptor have reduced numbers of ACh receptors(5). Muscle from human myasthenia gravis patients also aredeficient in ACh receptor (ref 14; Lindstrom and Lambert,unpublished data). Whether or not this observed deficiency inreceptor density found in vivo is due to modulation is underinvestigation.The finding that antibody against the ACh receptor induces

a reduction in the density of ACh receptors is similar to thephenomenon of antigenic modulation found in other systems,for example H-2 antigen, TL antigen, and surface immuno-globulins (31-33). The densities of hormone receptors [insulin(34), thyrotropin-releasing hormone (35)] and the fl-adrenergicneurotransmitter receptor (36, 37) are modulated by specificreceptor ligands. It seems plausible to us that, in addition toantibody, other ligands will be found that regulate the densityof ACh receptor on muscle.

We thank Dr. Jim Patrick for many discussions, providing data onthe BC3H-1 cell line prior to publication, and providing 125I-a-BuTx.We thank Dr. V. Lennon for providing immune sera. We thank B.Carlisle and J. Clark for technical assistance. This study was supportedby grants from the Muscular Dystrophy Associations of America to S.H.and J.L. and the National Institutes of Health (1-R01 NS11549 and3-R01 NS11323). S.H. was supported by a National Institutes of HealthResearch Career Development Award (5-K04 NS70139-04), S.B. bya Muscular Dystrophy Associations of America Fellowship, and R.K.by a Canadian MRC Fellowship. J.R. was supported by NationalScience Foundation Grant MPS 75-08003.The costs of publication of this article were defrayed in part by the

payment of page charges from funds made available to support theresearch which is the subject of the article. This article must thereforebe hereby marked "advertisement" in accordance with 18 U. S. C.§1734 solely to indicate this fact.

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