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ORIGINAL ARTICLES

Evidence forcquired Neuromyotonia:Autoantibodies Directed Against K+Channels of Peripheral NervesPaul Shillito, MRCP, ' Peter C. Molenaar, Ph D, t Angela Vincent, MRCPath, Katherine Leys, DPhil,

Wang Zheng, MSc,l Rutgeris J. van den Berg, PhD,t Jaap J. Plomp, MSc,iGertrudis Th. H. Van Kempen, BScJ Guy Chauplannaz, Axel R. Wintten, MD,SJ. Gert van Dijk, MD,S and John Newsom-Davis, FRS

Acquired neuromyotonia is characterized by hyperexcitability of motor nerves leading to muscle twitching, cramps,and weakness. The symptoms may improve following plasma exchange, and injection of immunoglobulin G 1 6 )from neuromyotonia patient into mice increased the resistance of neuromuscular transmission to d-tubocurarine.Here we examine nerves and muscle in vitro from mice injected with plasma or purified IgG from 6 neuromyotoniapatients or pooled control subjects, and cultured dorsal root ganglion cells after treatment with IgG. Three of thepatients had antibodies against human voltage-gated potassium channels labeled with ' 251-~-dendro tox in.he quantalrelease of acetylcholine (quantal content) at end-plates in diaphragms from mice treated with neuromyotonia IgGpreparations was increased by 21 relative to control values p = 0.0053 .With one IgG preparation, the durationof the superficial peroneal nerve compound action currents was increased by 93 . The dorsal root ganglion cellstreated with this IgG showed a marked increase in repetitive firing of action potentials. All effects were similar tothose obtained with aminopyridines. We conclude that at least some patients with acquired neuromyotonia haveantibodies directed against aminopyridine- or a-dendrotoxin-sensitive K + channels in motor and sensory neurons,and they are likely to be implicated in the disease process.

Shillito P, Molenaar PC, Vincent A, Leys K, Zheng W, van den Berg RJ, Plomp JJ, Van Kernpen GThH,Chauplannaz G, Wintzen AR, van Dijk JG, Newsom-DavisJ. Acquired neuromyotonia: evidence for

autoantibodies directed against K+ channels of peripheral nerves. Ann Neurol 1995;38:714-722

Neuromyotonia (NMT) iri characterized clinically bymuscle twitching during rest (myokymia), cramps, es-pecially induced by muscle contraction, impaired mus-cle relaxation, and sometimes muscle weakness; in-creased sweating; and a raised creatine kinase level 111.The clinical syndrome has been variously described asundulating myokymia 121, continuous muscle fiber ac-tivity [ 3 } and NMT [4}-the designation used here. I tmay have a known precipitating cause (e.g., hereditaryneuropathy) or occur as an acquired disorder with orwithout evidence of an associated neuropathy. This ar-ticle concerns acquired NMT.

Isaacs [3] established the peripheral nerve origin ofNMT by showing a persistence of abnormal electro-myographic (EMG) activity after proximal motor nerveblock and its disappearance after curarization; he sug-gested that the abnormal activity arose in the terminalarborizations of motor nerves in his patients. Subse-quent studies showed that in some patients there maybe more proximal sites of impulse generation in themotor nerve 15-81. Clinical features suggesting that

immunological mechanisms may be involved in the eti-ology of NMT include increased association with myas-thenia gravis or thymoma and raised anti-acetylcholine(ACh) receptor antibody titers in some patients 19, 101,induction by penicillamine 1111, and the presence ofcerebrospinal fluid oligoclonal bands [S]. The responseto plasma exchange 112, 131 and passive transfer stud-ies 1121 points to a circulating factor that is likely tobe an antibody. Injection of NMT plasma or IgG intomice caused an in vitro resistance of the phrenic nerve-diaphragm preparation to tubocurarine [121, consistentwith an antibody-induced facilitation of neuromusculartransmission.

Here we have pooled results from laboratories inOxford and Leiden. We each used microelectrode elec-trophysiological methods to investigate the mechanismof the resistance to tubocurarine in diaphragms frommice injected with NMT IgG. In addition, in Leidenthe compound action currents of the superficial pero-neal nerve of the mice and the effect of NMT IgG ondorsal root ganglion (DRG) cells in tissue culture were

From the 'Neurosciences Group, Institute of Molecular M edicine,Universitv of Oxford, United Kingdom; De partm ents of tPhysiol- Received Mar 20, 1995,and in revised form Jun 9 and 30. Acceptedfor publication Jul 3, 1995.om and $Neurology, Universiry ofleiden,Leiden, Netherlands; andQHBpital Neur ologique, Universite Cla ude Bernar d, Lyon, France.

Address to Dr Vincent, Insticute of M olecular Med-icine, Jo hn Radcliffe Hospital, O xford, O X 3 9DU United Kingdom.

714 Copyright 1995 by the American Neurological Association

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Table 1. Clinical Features o Patients with NeuromyotoniaPatient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6

SexAge at onset (yr)Duration (yr)Visible myokymiaCrampsPseudomyotoniaInreased sweatingEMG burst dischargesMaximum intraburst frequency (per sec)Effect of PEPeripheral nerve conductionAutoantibodies

Anti-VGKC 1251-a-dendrotoxin-VGKC(PM)

M57

0.5

++170NNoneJ

5

M17

7+tt

120NThyroidA N ASmooth muscle890

.1

F650.2+++150MF.1G M ,

161

M439+++50AxN one?1

10

~ F47

3++100N DS-MN one

49

F39

5++250NA N A1

247~~

PE = plasma exchange; + = present; = absent; = decrease; N = normal; MF = multifocal neuropathy; Ax = zo na l neuropathy;S-M = sensorimotor neuropathy; ND = not determined; EMG = electromyography;VGKC = voltage-gated K +channel; ANA = antinuclearantibody.studied. In each case we compared the results with thoseof the potassium channel blockers 4-aminopyridine(4A P) and 3,4-diaminopyridine (3,4D AP). Som e of theresults were briefly reported previously C141.Materials and MethodsClinical MaterialTable 1 summ arizes the clinical and e lectrophysio logical fea-tures of the 6 patients with NMT whose plasma or IgG wasused in these studies (Patients 1-5 in Oxford, Patient 6 inLeiden). Clinical features of Patients 1, 2, 4, and 5 weredescribed previously [S, 131. All except Patient 1 had myo-kymia (visible muscle twitching), but Patient 1 had otherfeatures associated with NMT (muscle cramps, pseudomyo-tonia, raised creatine k inase level). No ne had a known precip-itating cause for NMT. Plasma and serum from Patient 3(described in 1131) were from a case described by the lateprofessor S. Bady. The EMG features of NM T were presentin all patients. Th ese consisted of do ublet, triplet, or multi-plet bursts of repetitive firing of the same motor unit, withmaximal intraburst frequencies of 50 to 250 Hz. The burststhemselves occurred either regularly or irregularly, de-pending on the patients, at intervals of 1 to 30 seconds.Fibrillation potentials, fasciculations,or both were also pres-ent in Patients 1 , 2, 4 , and 5 .Patients 1, 2, and 6 had no evidence of other neurologicaldisease. Patient 3 presented with subacute sensory motorneurop athy. Clinical exam ination of Patients 4 and 5 wasunremark able, but nerve co nduction studies revealed an axo-nal type of peripheral sensory neuropathy in Patient 4 and amild sensory neuropathy in Patient 5 .The patients had received varying benefit from standardtreatment for N M T (phenytoin and carbamazepine). Thes ehad been of only limited value in Patients 2 and 4 whoseNM T sym ptoms were a cause of major disability preventingnormal employment. Patients 1 through 4 and 6 had under-

gon e therapeutic plasma exchange and the responses for Pa-tients l , 2, 4 , and 6 have been reported elsewhere [S, 13,153.Assay of Anti-K' Chann el AntibodySera were tested for antibodies to voltage-gated K + channels(VGKC) by immunoprecipitation of '251-a-dendrotoxin(DnT x)-labeled extracts of human frontal cortex as will bedescribed in detail elsewhere I . Hart, C. Waters, K. Leys,e t al, manuscript in preparation). Briefly, frontal cortex wasobtained postmortem and the membranes extracted for 30minutes in 1 digitonin as previously described for cerebel-lar cortex [16]. '251-a-DnT x (2,0 00 Ci/mmol) was obtainedfrom Amersham International.Aliquots of digitonin extract, 25 p.1, were added to 6.5fmol Iz5I-a-DnT x 2 0,000 cpm) in 25 p.1of PTX buffer (0.02M phosphate, pH 7.2, 0.1% Triton X-100). Serum(1.25-10.00 pl diluted 1 : l O in F'TX) was added and incu-bated overnight at 4C. Goat antihuman IgG was added inexcess and the precipitate centrifuged at 5,000 rpm for 5minutes. The precipitates were washed in F'TX three timeswithout resuspension and counted on a Packard GammaCounter. Parallel assays were performed in the presence ofexcess cold a-DnTx to block specific binding. Results aregiven as picomoles (pM) of ' I-DnTx binding sites per literof seru m after subtraction of nonspecific binding.Passive Im munization of MiceN M T plasma was obtained by plasma exchange, and controlplasma from a pool of blood donors. IgG was extracted fromplasma using the ethacridine (Rivanol)/ammonium sulfatemethod [17). The final IgG concentration of each prepara-tion was measured by an enzyme-linked immunosorbentassay (ELISA). Th e I gG from Patient 6 and the pooled con-trol IgG fractions were purified by affinity chromatographyI l S I .

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MF 1 and Sw iss mice (20-25 gm oth sexes), respectively,were used for the experiments in Oxford and Leiden. Themice received daily intraperitoneal injections of either 17 to22 mg of 1gG (10-13 days, Patients 1-5 and controls) or 1ml of plasma (14-30 days, Patien t 6 and relevant controls).O n day 2, cyclophosphamide (30 0 mglkg) was given to sup-press the immune response to the injected IgG.The mice were killed by cervical dislocation and the leftphrenic nerve-hemidiaphragm was dissected. In th e experi-ments performed with the material of Patient 6, the extensordigitorum longus (EDL), the soleus muscle, and the peronealnerve were also dissected see below).Electrophysiology o NeurotrunsmitterAcetylcholine ReleaseMuscles were mounted in a bath continuously perfused withoxygenated (9 5r r oxygen/5% carbon dioxide) medium at 23to 28C. In the experiments in Oxford, the medium wasof the following composition (mM ):sodiu m chloride (NaCI),118 ; potassium ch loride (KC I), 4.7; magnesium sulfate(M gS 04 ), 1.2; calcium ch loride (CaCI,), 2 .52; sodium bicar-bonate (Na HC O, ) , 25; glucose, 11.1; p H 7.4. In the experi-ments in Leiden the medium was a Ringer's solution con-taining NaC1,11 6 mM; KCI, 4.5 mM; MgSO, , 1 mM; CaCI,,2 mM; NaH ,PO 4, 1 mM; NaH CO , , 23 mM; and glucose ,11 mM, p H 7 .4 .Intracellular voltage recordings of miniature end-plate po-tentials (MEPPs) and end-plate potentials (EPPs) were madefrom the end-plate region of muscle fibers, using standardtechniques. Recordings w ere only ma de from fibers with rest-ing membrane potentials more negative than -60 mV. Atleast 20 MEPPs and EPPs were recorded from each fiber.To be able to meas ure EPPs, the nerve-evoked muscle actionpotential was blocked by treatment for 40 to 60 minutes with2.3 pM pc on ot ox in G III B (Scientific Marketing Associates,Barnet, United Kingdom), which blocks voltage-gated Na+channels of mouse muscle but not, at this concentration,those of nerve [19], and doe:; not influence the quantal re-lease of ACh and its effect on ACh receptors 120, 211. Forthe generation of th e EPPs th e phrenic nerve was stimulatedat 1 or 0.3 sec-I . T he amplitudes of the MEPPs and EPPswere normalized to 5 mV , assuming 0 mV as the reversalpotential for the ACh-indu ced current 1221. Th e normalizedEPPs were corrected for nonlinear summ ation with th e for-mula of McLachlan and Martin 1231 using a value for f of0.8.Monophasic Recording o Netwe Action CuwentsTh e mous e sciatic nerve was dissected from its entrance intothe leg. All nerve branches were cut except the superficialperoneal nerve, which was dissected to near the ankle. Th enerve was carefully desheath ed un der the dissection micro-scope and t he p eroneal n erve part o f the sciatic was cleavedfrom the rest in a retrograde direction. T he nerve was keptand tested in th e following med ium (mM ): NaCI, 136; KCI,4 .6 ; MgCI, , 1 ; CaCI,, 2 ; N aH ,P 0 4 , 1; NaHCO,, 2.5; glu-cose, 11 ; and Tris buffer, 5, p H 7.4. Stimulation and record-ings of nerve action currents were performed in a five-comp artmen t chamber in which t he nerve was covered withpetroleum jelly between the different compartments for elec-trical insulation 1241. Action currents at the ends of the

nerves, in the outer compartments, were abolished in me-dium with 13 6 mM choline chloride instead of N aCl 1241.Action Potentials and Cuwents in Dorsal RootGanglion CellsD R G from newborn Wistar rats were dissociated, plated,and cultured in Dulbecco's modified Eagle medium (DM EM )with 10% calf serum as described in detail elsewhere 1251.D R G neurons were cultured in the presence of 2 mg ml - 'of IgG from Patient 6 or control I&. In paired experim entsthe DRG cells were cultured for about 22 hours in the ab-sence of IgG and then for 2 hou rs in its presence. D R G cellsshowed little outgrowth up to 24 hours of plating. Theirexcitation was investigated using the patch-clamp pipette inthe cell-attached or whole-cell mode {25].StatisticsThe values are presented as the mean standard error ofth e mea n (S EM ). Possible statistical differences we re ana-lyzed with the unpaired Student's t test unless indicated o th-erwise. In the experiments in which the effect of IgG injec-tions was tested , two-tailed p values were calculated on thebasis of the mean values of the data obtained from eachmouse (n = num ber o f mice).ResultsAntibodies to 12 I-a-Dendrotoxin-labeled PotassiumChannels in Patients with NeuromyotoniaW e looked for antibodies to VGKCs in sera fromN M T patients and controls using as antigen >I-a-DnTX-labeled VG KC s extracted from human cortex.Patients 2 and 6 had titers of 890 and 247 pmol (seeTable 1). Patient 3 showed low positive values (mean,161 pmol), just above the mean + 3 standard devia-tions (SDs) (20 4 3 pmol, n = 1 7 ) for controls,which included healthy individuals and patients withLambert-Eaton myasthenic syndrome or myastheniagravis. The remaining 3 patients did not have raisedanti-VGKC antibodies.Antibodies against the ACh receptor were not de-tected by routine immunoprecipitation (values < 0.2nM a-bungar otoxin-bin ding sites) in any of the patients(see 19, 101).Response to Plasma Exchange andImmunosuppressive TreatmentPatients 1, 2 , 3 , and 6 showed subjective and EMGevidenc e of improv emen t following therapeutic plasmaexchange IS, 12, 13, 151. Serial serum samples wereavailable from Patient 2. Anti-VGKC antibody titersdeclined following plasma exchange and associatedtreatment with prednisolone and azathioprine (Fig 1).At the end of the treatment period, myokymia wasscarcely evident clinically, and the EMG showed alower level of spontaneous activity. As a result, thepatient elected to stop the treatment, which was laterfollowed by clinical relapse.

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1001900-800700-600500-400-300-200-1004

Plasma exchangen

IPredkaI

-10 0 10 20 30 40 50 60Time (months)

F i g 1 . Anti-voltage-gated Kf channel (VGKC)antibodies inPatient 2 before and after plasma exchange and following immu-nosuppressive therapy (PredIAta). Results are mean ? SEM othree independent assays.Passive Transfer Experiments in MiceThe effect of IgG or plasma from Patients 1 to 6 wasexamined after daily injections for 10 to 30 days andcompared with the effect in uninjected mice or miceinjected with control plasma. None of the 26 miceinjected with the IgG obtained from the patientsshowed signs of N M T clinically. In addition, neith erthe diaphragm nor the EDL or soleus muscles showedspontaneous contractions in vitro.Neuromuscukzr Junctio nTable 2 shows the mean results from th e electrophysio-logical measurements for N M T and control plasma-

treated mice. Values for resting membrane potential,MEPP amplitude and frequency, and t , /*of EPPs didnot differ significantly between the two groups. TheMEPP amplitudes of diaphragms from mice treatedwith material from Patient 1, 3 , or 6 were a littlesmaller than contro l amplitudes, bu t this difference wasnot statistically significant.The amplitude of the EPPs was larger in the test(Patients 1-5) than in the control diaphragms ( p =

0.017). N o spontaneous or repetitive EPPs were ob-served. Figure 2 shows the quantal contents of the EPPin test and control mice. In each case, test values werehigher than control values, and the differences weresignificant for mice receiving material from Patient 1or 6 ( p = 0 .0 27 a n d p = 0.014). All quantal contentswere h igher in the diaphragms of the Leiden mice (Pa-tient 6) than those in Oxford (Patients l-5), perhapsdu e to the different mouse strains used and small dif-ferences in the experimental conditions. When resultsof NMT-treated diaphragms were expressed as a per-centage of th e relevant controls, the mean values weresignificantly raised ( 12 1 596 mean ? SEM; n = 6patients; p = 0.0053, paired t test).To see whether the effects observed o n quantal con-tent might be related to a change in the number offunctioning potassium channels, we used the potassiumchannel blockers 4A P and 3,4DAP . A t concentrationsbetween 1 and 100 pM, 4AP had no effect o n theresting membrane potential of the mouse phrenicnerve-hemidiaphragm preparation. Th e amplitude andtime course of th e MEPPs were also unaffected. How -ever, addition of l pM 4A P increased the quantal con-tent from 29.6 & 2.7 to 36.8 ? 3.1 ( p = 0.05); at

Table 2. Efiect o Passive Transfe r o Neuromyotonia ( N M T ) IgG or N M T Plasma on the Diaphragm o the MouJea~~~~~~~ ~ ~ ~~

EPP MEPPSource of IgGor Plasma n RMP (mV) Amplitudeb (mV) tl,: (msec) Amplitude' (mV) Frequency (sec-')Patient 1Patient 2Patient 3Patient 4Patient 5MeaneC o n t r o l s ePatient 6'Control'

4 -75 1.7 23 1.34 -73 1.3 25 1.54 -73 ? 2.1 21 ? 2.24 - 7 2 1.6 23 1.54 -77 1.0 24 ? 2.0

20 - 7 4 a 0.7 23 ? 0.8'18 -72 0.9 21 0.66 -74 1.5 31 ? 0.76 -75 0.8 29 1.4

1.6 0.15 0.9 0.042.2 0.20 1.0 ? 0.062.2 0.30 0.9 0.132.0 ? 0.20 1.1 0.081.6 ? 0.15 1.1 ? 0.081.9 0.07 1.0 ? 0.041.9 ? 0.09 1.0 0.042.0 0.08 0.8 ? 0.032.0 0.06 0.9 0.04

1 .1 0.130.9 ? 0.191.0 0.220.7 0.100.8 0.120.9 0.030.9 0.041.0 0.071.0 0.07

~ ~~ ~ ~~

'Mice were treated with Ig G obtained from patients 1-5 or with plasma from Patient 6; n = num ber of mice, 5-1 5 end-plates were investigatedper muscle.bEPPs not yet corrected for nonlinear summation.'MEPPs and EPPs normalized to 5 mV RMP.dTime elapsed from peak of EPP to 50%.'EPPs recorded at 1 sec-' stimulation.'EPPs recor ded at 0.3 sec-' stimulation.gDifferent from control, p 2 = 0.017.EPP = end-plate potential; MEPP = miniature end-plate potential; RMP = resting membrane potential.

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3 r

60O I- 5CU 40-

306M10

~~ NM Tl NMTP NMTB NMT4 NMTB NMTG

F i g 2. Eflect of treating mice with neuromyotonia (NMT)IgGor plasma (hatched columns) o n the qua ntal content o the dia-phragm compared with control IgG or plasma (open columns) .Experiments werr pevfomed in Oxford u'ith stimulation at 1sec-' (Patients 1-5) r in Leiden at 0.3 sec-' (Patient 6). Thenumber of mice is indicated i n parentheses; Patients 2 and 4shared the same control group of 6 micr. The large standard er-ror bar in Patient 3 was due t o tiuo values o high 58 and 46)and two i'alues o low (22 and 2'3) quanta1 content. Th e folloui-ing test results were statistically significantly different from con-trol values: N M T 1 p = O.O2?; N M T 6. p = 0 .014. NM T2 us controls was p = 0.06.

100 p M the quantal content was 52.2 9.2 ( p

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Table 3 . Effect o Pauive Transfer uith Neuromyotonia Plasmaon the Super ficial Peroneal Nerve o the Mouse'

Table 4 fiect o Neuromyotonia IgG on Dorsal Root Ganglion(D RG ) Cellsfrom the Rat in Tissue CultureDuration of the Test IgG Control I g GA aP Cur rent (msec) Resting membrane PO- - 5 8 3 (10) - 5 2 * 3 (12)Control Plasma/ tentials (m V)Test Plasma Untrea tedb

Nerve in Ringer 1.5 ? 0.33 (7)Nerve in Ringer + 13.5 * 2.8 5 ) 12.2 * 3.9 ( 7 )2.9 * 0.43 (5)'500 p M 3,4DAPMice were treated with plasma from Patient 6 or from blood donors.The width of the current was taken at 10% of i ts maximum. Th enumber of mice is in parentheses.bResults pooled from 4 mice injected with plasma and 3 untreatedmice.'Different from controls, p = 0.025.

3.4DAP = 3,4-diaminopyridine.to 3.0 0.49 msec (n = 5 , p = 0.010, paired t test).This effect proved rapidly reversible after washout of3,4DAP. At 500 pM, 3,4DAP caused an approxi-mately tenfold increase in the width of the AaP cur-rent (see lower panel of Fig 3 and Table 3). At thisconcentration of 3,4DAP, the current traces showed anoisy baseline and in som e nerves spont ane ous individ-ual small action currents could be distinguished. Thenoise was characterized in a power spectrum whichindicated dominant frequencies at 3 and 5 Hz. In afew nerves, when th e a ibers were stimulated at a lowstimulating voltage in the presence of 500 p M3,4D AP, repetitive activity could be recognized as in-dividual currents. Their frequency was extremely high(between 500 and 1,000 Hz .Effect o IgG on Dorsal Root Ganglion Cells inTissue CultureFinally, to see whether NMT IgG could affect K+channel function in vitro, we used tissue culture. Incu-bation of DRG cells for 24 hours at 37C in the pres-ence of 2 mg ml-I of IgG from Patient 6, comparedto incubation in the presence of 2 mg ml-' of controlIgG, did not influence the resting me mbr ane potential,the firing threshold, or the size of action potentials andtheir dura tion (T able 4).

Untreated or control IgG-treated D R G cellsshowed occasionally repetitive activity when investi-gated in the cell-attached patch configuration undervoltage-clamp mode or whole-cell patch configurationund er current-clamp conditions (Table 4, Fig 4). Whe nthe cells had been treated with the IgG from Patient6, repetitive activity was increased from about 10 to70% (cell-attached mode) or from about 30 to 70(whole-cell mode). In two cells in the cell-attachedpatch configuration, spontan eous action curren ts wereobserved. S pon tane ous and increased repetitive activ-ity was not found when test cells were incubated for

Action potentialsThreshold (mV)Overshootb (mV)Width' (msec)

Repetitive firingdCell-attached patchWhole-cell patch

- 2 0 4 ( 7 )4 2 7 (7)4 l ( 7 )

9113'517'

-18 -+ 4 (7)37 * 6 (7)5 -+ l ( 7 )

1/133/9g; 2 1 / 8 0 hSpontaneous firingdCell-attached patch 21 13 0113'DR G cells were incubated for 24 hours at 37C in culture mediumin the presence of IgG from Patient 6 or controls. Th e number ofcells is in parentheses.bOv ersh oot of the action potential above 0 mV.'The width of the action poential was taken at 50% of its maximum.'Nu mb er of cells showing repetitive (spontaneous) firing/total num-ber of cells investigated.eStatisticaUy differe nt fro m c ontro ls ( p = 0.004).p = 0.046, test vs control and untreated combined: Fisher's 2 x 2exact test.K el l s treated with control IgG.hCells treated without IgG.

only 2 hours in the presence of 2 mg ml-I of IgGfrom Patient 6 (not illustrated).Untreated DRG cells, when exposed to 500 p M3,4DAP, also showed frequent repetitive activity un-der whole-cell current-clamp conditions (see Fig 4).However, no spontaneous currents were observed inthe presence of 3,4DAP.DiscussionEvidence far Impaired K+ Channel Function andInvolvement ofAnti-VGKC AntibodiesThis report presents converging evidence consistentwith the hypothesis that blockade of VGK Cs, or a de-crease of their number, can be an underlying causeof N M T. Th e unequivocal evidence of antibodies toVGKC in 2 of the patients, the passive transfer studiesin mice, the in vitro effect of purified IgG o n D R Gcells, and the similarity between the results and thoseof low doses of the potassium channel blockers 4APand 3,4 DA P strongly support the notion that one formof acquired N M T is autoimm une and that anti-VGKCantibodies play a part. If this proves to be the case,N M T c an be added to myasthenia gravis and the Lam-bert-Eaton myasthenic syndrome as examples of disor-ders in which ion channels are specifically targeted byautoantibodies. T he mechanisms by which the IgG an-tibodies act are not yet clear; the lack of effect onD R G cells when th e incubation was limited to 2 hourssuggests that in that situation, a dire ct block of channelfunction is not involved.

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40200

-20-4060

>

0 100 200 300 400 50080S

control IgG

~

A

-20-4060

>E

6o Icontrol:I

100 200 300 400 500ms

-80

C

8o I NMT IgG60 -4020 I I I80 I I I I0 100 200 300 400 500

msB6o I 3.4OAP

-80 L0 100 200 300 400 500

msDFig 4. Representative trace1 of action potentials of rat dorsal rootganglion cells in tissue culture, measured in the whole-cell patchmode under current clamp, after incubation for 24 hours in thepresence o 2 mg ml-' o control or Patient 6 IgG. There was re-petitive dctiuity in the neuromyotonia (NMT) IgG-treated B)but not in a control-treated cell (A). Repetitive action potentialsalso occurred in the presence ( D ) but not in the absence C ) of500 l 3,4-diaminopyridine(3,4 DAP).

Spec$city of Radioimmunoassc;cyfor Anti-VGKCAntibodiesVGKCs derive from alternate splicing of many differ-ent VGKC gene products. 3,4DAP and 4AP act onseveral types, but a-DnTx binds to a limited numberof forms only. For example, a-DnTX is ineffective inmuscle cells 1271, but it increases ACh release byblocking Kf channels of nerve terminals 128) andblocks a fast activating type of K+ channel in the nodesof Ranvier of motor nerves 1129-311. The results fromthe radioimmunoassay with lZSI-a-DnTx-labeled brainVGKCs were above the control range in Patients 2, 3 ,and 6, indicating the presence of antibodies to thesechannels. Results from the remaining 3 patients werenot different from control results. The skewing of theNMT results toward values within the control rangesuggests that the assay lacks the sensitivity to detectantibodies in all patients. Moreover, using a new, im-munohistochemical approach we obtained positive re-sults in a high proportion of NMT patients usingrecombinant VGKC subtypes (HBK2 and HBK5;I. Hart, C. Waters, A. Vincent, J. Newsom-Davis,unpublished observations, 1995).

Effects at the Neuromusculur JunctionThree of the 6 sera from the patients caused an in-crease of the mean quantal content of end-plates of themouse diaphragm. These results were consistent withprevious evidence that plasma and IgG from Patient 2,injected into mice, increased the efficiency of neuro-muscular transmission in vitro, as shown by the in-creased resistance to the paralysis caused by tubocura-rine [12]. The MEPP amplitudes in mice injected withIgG or plasma from NMT Patients 1, 3, and 6 wereslightly decreased; this might have been caused by thepresence of antibodies to the ACh receptor, sinceanti-ACh receptor antibodies are sometimes found inNMT patients [9, lo}, but none of these patients werepositive for anti-AChR.In the 3 patients with neuropathy (Patients 3-5)some of the clinical symptoms could have arisen as asecondary result of peripheral nerve damage. Nerve-induced continuous muscle fiber activity, which is thehallmark of NMT, could, in principle, be the result ofinhibition of acetylcholinesterase (AChE) at the end-plate {32}, or due to abnormal or prolonged activityof Na' channels at the nodes of Ranvier. The latterwould be difficult to distinguish electrophysiologicallyfrom a block of VGKCs. However, the activity ofAChE at end-plates of mice treated with Patient 6plasma was not reduced (P. Molenaar, G. van Kempen,unpublished observations, 1994). In Patient 3, in addi-tion to a low level of anti-VGKC, other autoantibodiesincluding anti-GM and antinuclear antibody werepresent, consistent with an autoimmune process. How-

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ever, Patients 4 and 5 had no evidence of autoimmu-nity and the response to plasma exchange in Patient 4was unclear. In these patients, therefore, the experi-mental evidence for a role of antibodies is not yet con-clusive.Effects at the NeweIt might be expected that the injected human IgG inthe mouse had more easy access to the motor nerveterminals than to nodal and paranodal regions in thenerve. However, the fact that action currents in thesuperficial perone al nerv e were m arkedly prolongedafter treatment with Patient 6 plasma indicates that theblood-nerve barrier was breached to some extent,though neuromyotonia-like symptoms were not seenin the m ice. Presumably the proportion of ion channelswhose activity was reduced was not sufficient to causefrequent spontaneous or repetitive activity in motornerves. By contrast, the experiments with the DRGcells showed that when nerve cells were directly ex-posed to NM T I&, or to 3,4DAP, repetitive activitydid indeed occur in vitro. In this connection it is ofinterest that a-DnTx is a selective blocker of a rapidlyactivating, sustained K f current of a low conductanceK + channel in D R G neurons {33-351 causing repeti-tive firing of action potentials. These channels wouldappear to be a likely target for anti-VGKC antibodiesin these cells.At the frog node of Ranvier the number of K + chan-nels is relatively scarce compared to the number ofN af channels; in mammalian nerve the number of K Cchannels is even smaller [36]. Therefore, it is some-what surprising that 3,4DAP, and a-DnTx in experi-ments by others caused repetitive action potentials inthe peroneal nerve of the mouse and the DRG cellsof the rat. Earlier reports on sciatic nerve from youngand adult rabbits indicated that 4AP causes a prolonga-tion of the compound action potential in young rats,but n ot in adult animals {37-391. T h e effects of a-DnTx and 3,4DAP on the nerve strongly suggest thatspontaneous and repetitive movements in NMT aregenerated in the region of the no de of Ranvier througha decrease of the number of available VGKCs.

Th e symptoms of N M T have some resemblance tothe spontaneous movements of the ShakerDrosophikz,which carries a mutant gene for a VG KC {40]. Mousemutants of potassium channel genes that have beendescribed display head waving, body tremors, uncoor-dinated gait, and hyperactivity C4 I]. Recently, episodicataxia, a syndrome in which patients have attacks ofgeneralized ataxia and also experience myokymia dueto discharges in peripheral motor nerves, has been as-sociated with point mutations in the potassium channelgene KCNA1 {42]. This hereditary form of NMT ap-pears to involve a change of function rather than acomplete loss of one type of K + channel. O ur present

findings indicate that autoantibodies to VGKCs arepresent in some patients with NMT, and could be re-sponsible for their disordered function.W e gratefully acknowledge suppor t by the Medical Research Counciland Muscular Dystrophy Grou p of Great Britain, the Sir Jules ThornTrust, and the Nederlandse Organisatie voor WetenschappelijkOnderzoek (to P. C. M.).We thank D r An neke Brand for helpful discussions, and Ms Mo-nique van Lint for the purification of IgG.

References1. IG mura J. Neuromuscular diseases characterized by abnorm almuscle activity. In: E lectrodiagnosis in diseases of nerve andmuscle. Principles and practice. Philadelphia: Davis, 1983:549-5652. Denny-Brow n D , Foley JM. M yokymia and the benign fascicula-tion of muscular cramps. Trans Assoc Am Physicians 1948;61:88-963. Isaacs H.A syndrome of continuous muscle-fibre activity.J Neurol Neurosurg Psychiatry 1961;24:319-325

4. Mertens H G , Zschocke S. Neuromyotonie. IUin Wochenschr5 . Partanen VSG, Soininen H, Saksa M, Riekkinen P. Electromyo-graphic and nerve conduction findings in a patient with neuro-rnyotonia, norrnocalcemic tetany and small-cell lung cancer. ActaNeurol Scand 1980;61:216-2266. Roth G. Midmotoraxonal reexcitation in human peripheralnerve. Electromyogr Clin Neurophysiol 1985;25:401-4117. Hosokawa S, hinoda H, SakaiT , et al. Electrophysiologic studyon limb m yokymia in three women. J Neuro l N eurosurg Psychi-atry 1987;12 5:239-2468. Newsom-Davis J Mills KR. Immun ological associations of ac-quired neuromyotonia (Isaacs syndrome): repo rt of 5 cases andliterature review. Brain 1993;116:453-4699. Halbach M, Homberg V, Freund H-J. Neuromuscular, auto-nomic and central cholinergic hyperactivity associated with thy-rnoma and acetylcholine receptor-binding antibody. J Neurol

10. Garcia-Merino A, Cabella A, Mora JS, Lia6o H. Continuousmuscle fiber activity, peripheral neuropathy, and thymom a. AnnNeu rol 1991;29:2 15-2 181. Reeback J, Benton S, Swash M, Schwartz MS. Penicillamine-induced neuromyotonia. BMJ 1979;279:1464-14652. Sinha S,Newsom-DavisJ, Mills K, et al. Autoimmune aetiologyfor acquired neuromyotonia (Isaacs syndrome). Lancet 1991;

3 . Bady B, Chauplannaz G, Vial C, Savet J-F. Auto-im mune aetiol-ogy for acquired neu romyotonia (Isaacs synd rom e). Lancet1991;338:1330

4 Shillito P, I ang B, Newsom-Davis J, et al. Evidence for an auto-antibody mediated mechanism in acquired neuromyotonia.J Ne uro l Neuro surg Psychiatry 1992;55:12 145. Wintzen AR, Van Dijk JG, Brand A . Neu rom yoto nia with earlyresponse to plasrnapheresis associated with proximal action my-oclonus with late response to plasmapheresis. Muscle Nerve

6. MotomuraM ohnston I, Lang B, et al. An improved diagnosticassay for Lambert-Eaton myasthenic syndrome. J Neurol Neur-surg Psychiatry 19 95;58:85-877. Horeshi J, Smetna R. T he isolation of gamma globulins fromblood serum by Rivanol. Acta Med Scand 1956;155:65-708. Akerstrom B, Brodin T , Reis K, Bjork L. Protein G, a powerfultool for binding and detection of monoclonal and polyclonalantibodies. J Immunol 1985;135:2589-2592

1965 43:9 17-925

1987;234:433-436

338175-77

1994;17 suppl 1):S221

Shillito et al: K+ Channels in Neuromyotonia 721

8/12/2019 ACVKC

9/9

19. Cruz LJ, Gray WR, Olivera BM, et al. Conus geographrrs toxinsthat discriminate between neuronal and muscle sodium chan-nels. J Biol Chem 1985;260:9280-9288

20. Ho ng SJ. Chang CC . Use of gcographutoxin I1 (p-conoroxin)for the study of neuromuscular transmission in the mouse. BrJ Pharmacol 1989;97:934-940

21. Plomp JJ, Van Kempen G Th H , Molenaar PC. Adaptation ofquantal content to decreased postsynaptic sensitivity at singleendplares in a-bunga rotoxin-tre ated rats. J Physiol (Lon d) 1992;

22. Magleby KL, Stevens CF. The effecr of voltage on the timecourse of end-plate currenrs. J Physiol (Lond) 1972;223:15 -171

23. MacLachlan EM, Martin AR. Non-linear summation of endplatepotentials in the frog and the mouse. J Physiol (Land) 1981;311;24. Van den Berg RJ, Versluys CAA, De Vos A, Voskuyl RA.Nerve fibre size related block o f action currents by phenytoinin mammalian nerve. Epilepsia 1994;35:1279-128825. Wang 2 Van den Berg RJ, Ypey DL. Resting mem brane pot en-tials and excitability at different regions of regenerating do rsal

roo t ganglion ne urons in culture. Neur oscienc e 1994;60:245-254

26. Katz B, Miledi R. Estimates of cluantal content during 'chemicalpotentiation' of transmitter rek ase . Proc R SOC Lond [Biol]27. Harvey AL, Rowan EG, Vatanpour H, et al. Potassium c hannel

toxins and transmitter release. Ann N Y Acad Sci 1994;710.1-1028. Anderson AJ, Harvey AL. Effects of the potassium channelblocking dendrotoxins o n acetylcholine release and m otor nerve

terminal activity. Br J Pharmacol 1988;93:215-22129. Benoit E. Dubois J-M. Toxin I from the snake Dendroaspi~ o / y

1epsi.rpolylepsis:a highly specific blocke r of one type of potassiumchannel in myelinated nerve fiber. Brain Res 1986;377:374-37730. Jonas P, Brau M E, Herm steine r M, Vogel W. Single channelrecording in myelinated nerve hbers reveal one type of N a chan-nel, but different K channels. Proc Natl Acad Sci USA 1989;86:

4581487-499

307-324

1979;205:369-378

7238-7242

31. Brau ME, Dreyer F, Jonas P, et al. A K' channel in Xen0pu.inerve fibres selectively blocked by bee and snake toxins: bindingand voltage clamp experiments. J Physiol (Lond ) 1991;420:365-38532. Masland RL,Wigton RS. Ne rve activity accompa nying fascic-ulation produced by prostigmine. J Neurophysiol 1940;3:269-27533. Penner R, Petersen M, Pierau FK, Dreyer F. Dendrotoxin: aselective blocker of a non-inactivating potassium current inguinea-pig dorsal root ganglion neurons. Pflugers Arch 1986;407:365-369

34. Sransfeld CE, Marsh SJ, Halliwell JV, Brown DA. 4-Aminopyridine and dendrotoxin induce repetitive firing in ratvisceral se nsory neu rons by blocking a slowly inactivating out-ward current. Neurosci Lett 1988;64:299-30435. Stansfeld CE. Feltz A. Dendrotoxin-sensitive Kt hannels indorsal root ganglion cells. Neurosci Lett 1988;93:49-55

36. Chiu SY, Ritchie JM. Evidence for the presence of potassiumchannels in nodal and internodal axonal membrane of mamma-lian myelinated fibres. Nature 1981;284:170-171

37. Ritchie JM . Sodium and p otassium channels in regenerating anddeveloping mammalian myelinated nerves. Proc R SOCLond[Biol) 1982;215:273-287

38. Kocsis JD, Ruiz JA, Waxman SG. Maturation of mammalianmyelinated fibres: changes in a ction potential characteristics fol-lowing 4-aminopyridine application. J Neurophysiol 1983;50:440-463

39. Bournaud R, Mallart A. Potassium cha nnel blockers and impulsepropagation in murine motor endplate disease. Muscle Nerve1987;10;1-5

40. Butler A, Wei AG, Baker K, Salkoff L. A family of putativepotassium channel genes in Drosophila. Science 1989;243:943-94 741. Lock LF, Gilbert DJ, Street VA, et al. Voltagegated potassiumchanne l genes are clustered in paralogous regions of thecpmouse genome. Genomics 1994;20:354-362

42 . Browne DL, Gancher ST, Nur t JG , et al. Episodic ataxidmyo-kymia syndrom e is associated with point mutations in the humanpotassium channel gene, KC NA l. Natu re Ge net 1994;8:136-140

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