ORIGINAL ARTICLES

Acquired Neuromyotonia: Evidence for Autoantibodies Directed Against K+ Channels of Peripheral Nerves
Paul Shillito, MRCP,"' Peter C. Molenaar, PhD,t Angela Vincent, MRCPath," Katherine Leys, DPhil," Wang Zheng, MSc,l Rutgeris J. van den Berg, PhD,t Jaap J. Plomp, MSc,i Gertrudis Th. H. Van Kempen, BScJ Guy Chauplannaz, Axel R. Wintten, MD,S J. 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 1 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 neuromyotonia patients or pooled control subjects, and cultured dorsal root ganglion cells after treatment with IgG. Three of the patients had antibodies against human voltage-gated potassium channels labeled with '251-~-dendrotoxin. The quantal release of acetylcholine (quantal content) at end-plates in diaphragms from mice treated with neuromyotonia IgG preparations was increased by 21% relative to control values ( p = 0.0053).With one IgG preparation, the duration of the superficial peroneal nerve compound action currents was increased by 93%. The dorsal root ganglion cells treated with this IgG showed a marked increase in repetitive firing of action potentials. All effects were similar to those obtained with aminopyridines. We conclude that at least some patients with acquired neuromyotonia have antibodies 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-Davis J. Acquired neuromyotonia: evidence for autoantibodies directed against K+ channels of peripheral nerves. Ann Neurol 1995;38:714-722 Neuromyotonia (NMT) iri characterized clinically by muscle twitching during rest (myokymia), cramps, especially induced by muscle contraction, impaired muscle relaxation, and sometimes muscle weakness; increased sweating; and a raised creatine kinase level 111. The clinical syndrome has been variously described as undulating myokymia 121, continuous muscle fiber activity [ 3 } , and NMT [4}-the designation used here. It may have a known precipitating cause (e.g., hereditary neuropathy) or occur as an acquired disorder with or without evidence of an associated neuropathy. This article concerns acquired NMT. Isaacs [3] established the peripheral nerve origin of NMT by showing a persistence of abnormal electromyographic (EMG) activity after proximal motor nerve block and its disappearance after curarization; he suggested that the abnormal activity arose in the terminal arborizations of motor nerves in his patients. Subsequent studies showed that in some patients there may be more proximal sites of impulse generation in the motor nerve 15-81. Clinical features suggesting that immunological mechanisms may be involved in the etiology of NMT include increased association with myasthenia gravis or thymoma and raised anti-acetylcholine (ACh) receptor antibody titers in some patients 19, 101, induction by penicillamine 1111, and the presence of cerebrospinal fluid oligoclonal bands [S]. The response to plasma exchange 112, 131 and passive transfer studies 1121 points to a circulating factor that is likely to be an antibody. Injection of NMT plasma or IgG into mice caused an in vitro resistance of the phrenic nervediaphragm preparation to tubocurarine [ 121, consistent with an antibody-induced facilitation of neuromuscular transmission. Here we have pooled results from laboratories in Oxford and Leiden. We each used microelectrode electrophysiological methods to investigate the mechanism of the resistance to tubocurarine in diaphragms from mice injected with NMT IgG. In addition, in Leiden the compound action currents of the superficial peroneal nerve of the mice and the effect of NMT IgG on dorsal root ganglion (DRG) cells in tissue culture were

From the 'Neurosciences Group, Institute of Molecular Medicine, Universitv of Oxford, United Kingdom; Departments of tPhysiolom and $Neurology, Universiry ofleiden, Leiden, Netherlands; and QHBpital Neurologique, Universite Claude Bernard, Lyon, France.

Received Mar 20, 1995, and in revised form Jun 9 and 30. Accepted for publication Jul 3, 1995. Address to Dr Vincent, Insticute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DU United Kingdom.

714 Copyright 0 1995 by the American Neurological Association

Table 1. Clinical Features of Patients with Neuromyotonia

Patient 1 Sex Age at onset (yr) Duration (yr) Visible myokymia Cramps Pseudomyotonia Inreased sweating EMG burst discharges Maximum intraburst frequency (per sec) Effect of PE Peripheral nerve conduction Autoantibodies M 57 0.5
-

Patient 2 M 17 7

Patient 3 F 65 0.2

Patient 4
~

Patient 5 F 47

Patient 6 F 39

M 43

+ +
-

+
t
t

+
-

+ +
150

+ +
-

+ +
-

+ +
-

+
50

170

120

J
N None

.1
N Thyroid ANA Smooth muscle 890

.1
MF GM, 161

?1
Ax None

100 ND S-M None

250

1
N ANA

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

10
~~

49

247

PE = plasma exchange; + = present; - = absent; & = decrease; N = normal; MF = multifocal neuropathy; Ax = zonal neuropathy; S-M = sensorimotor neuropathy; ND = not determined; EMG = electromyography; VGKC = voltage-gated K + channel; ANA = antinuclear antibody.

studied. In each case we compared the results with those of the potassium channel blockers 4-aminopyridine (4AP) and 3,4-diaminopyridine (3,4DAP). Some of the results were briefly reported previously C141.
Materials and Methods Clinical Material
Table 1 summarizes the clinical and electrophysiological features of the 6 patients with NMT whose plasma or IgG was used in these studies (Patients 1-5 in Oxford, Patient 6 in Leiden). Clinical features of Patients 1, 2, 4, and 5 were described previously [S, 131. All except Patient 1 had myokymia (visible muscle twitching), but Patient 1 had other features associated with NMT (muscle cramps, pseudomyotonia, raised creatine kinase level). None had a known precipitating cause for NMT. Plasma and serum from Patient 3 (described in 1131) were from a case described by the late professor S. Bady. The EMG features of NMT were present in all patients. These consisted of doublet, triplet, or multiplet bursts of repetitive firing of the same motor unit, with maximal intraburst frequencies of 50 to 250 Hz. The bursts themselves occurred either regularly or irregularly, depending on the patients, at intervals of 1 to 30 seconds. Fibrillation potentials, fasciculations, or both were also present in Patients 1, 2, 4, and 5 . Patients 1, 2, and 6 had no evidence of other neurological disease. Patient 3 presented with subacute sensory motor neuropathy. Clinical examination of Patients 4 and 5 was unremarkable, but nerve conduction studies revealed an axonal type of peripheral sensory neuropathy in Patient 4 and a mild sensory neuropathy in Patient 5 . The patients had received varying benefit from standard treatment for NMT (phenytoin and carbamazepine). These had been of only limited value in Patients 2 and 4 whose NMT symptoms were a cause of major disability preventing normal employment. Patients 1 through 4 and 6 had under-

gone therapeutic plasma exchange and the responses for Patients l, 2, 4 , and 6 have been reported elsewhere [S, 13,
153.

Assay of Anti-K'

Channel Antibody

Sera were tested for antibodies to voltage-gated K + channels (VGKC) by immunoprecipitation of '251-a-dendrotoxin (DnTx)-labeled extracts of human frontal cortex as will be described in detail elsewhere (I. Hart, C. Waters, K. Leys, et al, manuscript in preparation). Briefly, frontal cortex was obtained postmortem and the membranes extracted for 30 minutes in 1% digitonin as previously described for cerebellar cortex [16]. '251-a-DnTx (2,000 Ci/mmol) was obtained from Amersham International. Aliquots of digitonin extract, 25 p.1, were added to 6.5 fmol Iz5I-a-DnTx(20,000 cpm) in 25 p.1 of PTX buffer (0.02 M phosphate, p H 7.2, 0.1% Triton X-100). Serum (1.25-10.00 pl diluted 1 : l O in F'TX) was added and incubated overnight at 4C. Goat antihuman IgG was added in excess and the precipitate centrifuged at 5,000 rpm for 5 minutes. The precipitates were washed in F'TX three times without resuspension and counted on a Packard Gamma Counter. Parallel assays were performed in the presence of excess cold a-DnTx to block specific binding. Results are given as picomoles (pM) of '"I-DnTx binding sites per liter of serum after subtraction of nonspecific binding.

Passive Immunization of Mice

NMT plasma was obtained by plasma exchange, and control plasma from a pool of blood donors. IgG was extracted from plasma using the ethacridine (Rivanol)/ammonium sulfate method [17). The final IgG concentration of each preparation was measured by an enzyme-linked immunosorbent assay (ELISA). The IgG from Patient 6 and the pooled control IgG fractions were purified by affinity chromatography IlSI.

Shillito et al: K + Channels in Neuromyotonia

715

MF1 and Swiss mice (20-25 gm,both sexes), respectively, were used for the experiments in Oxford and Leiden. The mice received daily intraperitoneal injections of either 17 to 22 mg of 1gG (10-13 days, Patients 1-5 and controls) or 1 ml of plasma (14-30 days, Patient 6 and relevant controls). O n day 2, cyclophosphamide (300 mglkg) was given to suppress the immune response to the injected IgG. The mice were killed by cervical dislocation and the left phrenic nerve-hemidiaphragm was dissected. In the experiments performed with the material of Patient 6, the extensor digitorum longus (EDL), the soleus muscle, and the peroneal nerve were also dissected (see below).

nerves, in the outer compartments, were abolished in medium with 136 mM choline chloride instead of NaCl 1241.

Action Potentials and Cuwents in Dorsal Root Ganglion Cells

D R G from newborn Wistar rats were dissociated, plated, and cultured in Dulbecco's modified Eagle medium (DMEM) 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 experiments the D R G cells were cultured for about 22 hours in the absence of IgG and then for 2 hours in its presence. D R G cells showed little outgrowth up to 24 hours of plating. Their excitation was investigated using the patch-clamp pipette in the cell-attached or whole-cell mode {25].

Electrophysiology of Neurotrunsmitter Acetylcholine Release

Muscles were mounted in a bath continuously perfused with oxygenated (95rr oxygen/5% carbon dioxide) medium at 23 to 28C. In the experiments in Oxford, the medium was of the following composition (mM):sodium chloride (NaCI), 118; potassium chloride (KCI), 4.7; magnesium sulfate (MgS04), 1.2; calcium chloride (CaCI,), 2.52; sodium bicarbonate (NaHCO,), 25; glucose, 11.1; p H 7.4. In the experiments in Leiden the medium was a Ringer's solution containing NaC1,116 mM; KCI, 4.5 mM; MgSO, , 1 mM; CaCI,, 2 mM; NaH,PO4, 1 mM; NaHCO,, 23 mM; and glucose, 11 mM, p H 7.4. Intracellular voltage recordings of miniature end-plate potentials (MEPPs) and end-plate potentials (EPPs) were made from the end-plate region of muscle fibers, using standard techniques. Recordings were only made from fibers with resting membrane potentials more negative than -60 mV. At least 20 MEPPs and EPPs were recorded from each fiber. To be able to measure EPPs, the nerve-evoked muscle action potential was blocked by treatment for 40 to 60 minutes with 2.3 pM pconotoxin GIIIB (Scientific Marketing Associates, Barnet, United Kingdom), which blocks voltage-gated N a + channels of mouse muscle but not, at this concentration, those of nerve [19], and doe:; not influence the quantal release of ACh and its effect on ACh receptors 120, 211. For the generation of the EPPs the phrenic nerve was stimulated at 1 or 0.3 sec-I. The amplitudes of the MEPPs and EPPs were normalized to - 75 mV, assuming 0 mV as the reversal potential for the ACh-induced current 1221. The normalized EPPs were corrected for nonlinear summation with the formula of McLachlan and Martin 1231 using a value for f of 0.8.

Statistics
The values are presented as the mean 2 standard error of the mean (SEM). Possible statistical differences were analyzed with the unpaired Student's t test unless indicated otherwise. In the experiments in which the effect of IgG injections was tested, two-tailed p values were calculated on the basis of the mean values of the data obtained from each mouse (n = number of mice).

Results Antibodies to 12' I-a-Dendrotoxin-labeled Potassium Channels in Patients with Neuromyotonia We looked for antibodies to VGKCs in sera from N M T patients and controls using as antigen ">I-aDnTX-labeled VGKCs extracted from human cortex. Patients 2 and 6 had titers of 890 and 247 pmol (see Table 1). Patient 3 showed low positive values (mean, 161 pmol), just above the mean + 3 standard deviations (SDs) (20 ? 43 pmol, n = 17) for controls, which included healthy individuals and patients with Lambert-Eaton myasthenic syndrome or myasthenia gravis. The remaining 3 patients did not have raised anti-VGKC antibodies. Antibodies against the ACh receptor were not detected by routine immunoprecipitation (values < 0.2 nM a-bungarotoxin-binding sites) in any of the patients (see 19, 101).

Monophasic Recording of Netwe Action Cuwents

The mouse sciatic nerve was dissected from its entrance into the leg. All nerve branches were cut except the superficial peroneal nerve, which was dissected to near the ankle. The nerve was carefully desheathed under the dissection microscope and the peroneal nerve part of the sciatic was cleaved from the rest in a retrograde direction. The nerve was kept and tested in the following medium (mM): NaCI, 136; KCI, 4.6; MgCI,, 1; CaCI,, 2; N a H , P 0 4 , 1; NaHCO,, 2.5; glucose, 11; and Tris buffer, 5, p H 7.4. Stimulation and recordings of nerve action currents were performed in a fivecompartment chamber in which the nerve was covered with petroleum jelly between the different compartments for electrical insulation 1241. Action currents at the ends of the

Response to Plasma Exchange and Immunosuppressive Treatment Patients 1, 2 , 3, and 6 showed subjective and EMG evidence of improvement following therapeutic plasma exchange IS, 12, 13, 151. Serial serum samples were available from Patient 2. Anti-VGKC antibody titers declined following plasma exchange and associated treatment with prednisolone and azathioprine (Fig 1). At the end of the treatment period, myokymia was scarcely evident clinically, and the EMG showed a lower level of spontaneous activity. As a result, the patient elected to stop the treatment, which was later followed by clinical relapse.

716 Annals of Neurology Vol 38 No 5 November 1995

1001 900800700-

Plasma exchange n

600500400-

300-

2001004

Predka
i

I
-10

10

20

30

40

50

60

Time (months)

Fig 1. Anti-voltage-gated Kf channel (VGKC) antibodies in Patient 2 before and after plasma exchange and following immunosuppressive therapy (PredIAta). Results are mean ? SEM of three independent assays.

Passive Transfer Experiments in Mice The effect of IgG or plasma from Patients 1 to 6 was examined after daily injections for 10 to 30 days and compared with the effect in uninjected mice or mice injected with control plasma. None of the 26 mice injected with the IgG obtained from the patients showed signs of NMT clinically. In addition, neither the diaphragm nor the EDL or soleus muscles showed spontaneous contractions in vitro. Neuromuscukzr Junction

Table 2 shows the mean results from the electrophysiological measurements for NMT and control plasma-

treated mice. Values for resting membrane potential, MEPP amplitude and frequency, and t,/* of EPPs did not differ significantly between the two groups. The MEPP amplitudes of diaphragms from mice treated with material from Patient 1, 3 , or 6 were a little smaller than control amplitudes, but this difference was not 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 observed. Figure 2 shows the quantal contents of the EPP in test and control mice. In each case, test values were higher than control values, and the differences were significant for mice receiving material from Patient 1 or 6 ( p = 0.027 a n d p = 0.014). All quantal contents were higher in the diaphragms of the Leiden mice (Patient 6) than those in Oxford (Patients l-5), perhaps due to the different mouse strains used and small differences in the experimental conditions. When results of NMT-treated diaphragms were expressed as a percentage of the relevant controls, the mean values were significantly raised (121 5 596, mean ? SEM; n = 6 patients; p = 0.0053, paired t test). To see whether the effects observed on quantal content might be related to a change in the number of functioning potassium channels, we used the potassium channel blockers 4AP and 3,4DAP. At concentrations between 1 and 100 pM, 4AP had no effect on the resting membrane potential of the mouse phrenic nerve-hemidiaphragm preparation. The amplitude and time course of the MEPPs were also unaffected. However, addition of l pM 4AP increased the quantal content from 29.6 & 2.7 to 36.8 ? 3.1 ( p = 0.05); at

Table 2. Efiect of Passive Transfer of Neuromyotonia ( N M T ) IgG or N M T Plasma on the Diaphragm of the MouJea
~~~~~~~ ~ ~ ~~

EPP Source of IgG or Plasma Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Meane
Controlse

MEPP
tl,:

RMP (mV)
4 4 4 -75 f 1.7 -73 ? 1.3 -73 ? 2.1 - 7 2 f 1.6 -77 f 1.0 - 7 4 a 0.7 -72 f 0.9 -74 f 1.5 -75 f 0.8

Amplitudeb" (mV)

(msec)

Amplitude' (mV)

Frequency (sec-')

23 25
21 23 24 23 21 31 29

f f

4
4 20
18

Patient 6' Control'

6 6

1.3 1.5 ? 2.2 f 1.5 ? 2.0 ? 0.8' ? 0.6 ? 0.7 f 1.4

1.6 2.2 2.2 2.0 1.6 1.9 1.9 2.0 2.0

0.15 0.20 2 0.30 ? 0.20 ? 0.15 2 0.07 ? 0.09 2 0.08 f 0.06

f

0.9 2 0.04 1.0 ? 0.06 0.9 f 0.13 1.1 2 0.08

0.08 0.04 ? 0.04 ? 0.03 0.9 f 0.04
1.1 1.0 1.0 0.8
?

1.1 0.9
1.0

0.7 0.8 0.9 0.9

1.0 1.0
~ ~~

0.13 0.19 f 0.22 f 0.10 f 0.12 f 0.03

f
?
?

0.04

? ?

0.07 0.07
~ ~~

'Mice were treated with IgG obtained from patients 1-5 or with plasma from Patient 6; n = number of mice, 5-1 5 end-plates were investigated per muscle. bEPPs not yet corrected for nonlinear summation. 'MEPPs and EPPs normalized to - 75 mV RMP. dTime elapsed from peak of EPP to 50%. 'EPPs recorded at 1 sec-' stimulation. 'EPPs recorded at 0.3 sec-' stimulation. gDifferent from control, p2 = 0.017. EPP = end-plate potential; MEPP
=

miniature end-plate potential; RMP

resting membrane potential.

Shillito et al: K+ Channels in Neuromyotonia

717

3
'OI 60
50

=z
2

c
C

- control

test

U c

40

30

M
10

"

~~

NMTl

NMTP

NMTB

NMT4

NMTB

NMTG

Fig 2. Eflect of treating mice with neuromyotonia (NMT) IgG or plasma (hatched columns) o n the quantal content of the diaphragm compared with control IgG or plasma (open columns).

5
4

Experiments werr pevfomed in Oxford u'ith stimulation at 1 sec-' (Patients 1-5) o r in Leiden at 0.3 sec-' (Patient 6). The number of mice is indicated in parentheses; Patients 2 and 4 shared the same control group of 6 micr. The large standard error bar in Patient 3 was due to tiuo values of high (58 and 46) and two i'alues of low (22 and 2'3) quanta1 content. The follouiing test results were statistically significantly different from control values: N M T 1 . p = O.O2?; N M T 6. p = 0.014. N M T 2 us controls was p = 0.06.

3 2
1

-10 U M

3.4 DAP

-control

100 pM the quantal content was 52.2 2 9.2 ( p < 0.02). The decay time constant of the EPP was not significantly increased at 1 pM 4DAP or 3,4DAP. At 100 pM 3,4DAP, the decay of the EPPs was greatly increased, namely from about 2 msec to values exceeding 50 msec (see [26]). Under this condition repetitive firing occurred after a single nerve stimulus, and furthermore, the preparation showed spontaneous twitching. This effect was abolished by application of 5 pM tubocurarine to the medium, indicating that it was due to presynaptic hyperactivity.

- 500 pM
2

- control

3,4 DAP

0
-2
0
I

10

ms

20

C
Fig 3. Single action currents of superficialperoneal nenles from mice treated with Patient 6 o r control plasma (A). or from nerves of untreated mice in thr absence o r presence of 3,4diaminopyridine (3,4-DAP), 10 pM (B) o r 500 pM (C). The amplitudes of the traces were uariable, depending on the thickness of the nenie bundles used, and were therefore normalized with regard t o the peak amplitude in order t o facilitate judgment of the shape of the currents.

Supe&ciaL Pemneal NerzJe We looked at nerves isolated from mice injected with Patient 6 plasma. Superficial peroneal nerves did not show spontaneous action potentials, nor did they fire repetitively upon a single nerve stimulus. The action currents of the control nerve, shown in Figure 3, had a A a p component (upper panel) whose shape was similar to earlier observations in the suralis nerve of the rat [24]. However, the nerves originating from mice treated with Patient 6 plasma showed a marked prolongation at the position of the (3 wave. As shown in Table 3, the width of the A ( Ywave ~ was increased from 1.5 to 2.9 msec. In addition, at low stimulus strength, about 0.15 V, which stimulates predominantly (Y fibers, a prolongation was already observed in the test nerve (from 1.0 k 0.25 to 2.5 5 0.59 msec,) = 0.026, not illustrated). Whereas in control nerves the width of the

action current increased by a factor of two, through recruitment of p fibers, when the stimulus voltage was increased from 0.16 to 0.50 V, such an increase was not found in the test nerves, probably because the increase of the width of the c t fibers overshadowed the increase in width caused by the recruitment of the p fibers. The middle panel of Figure 3 shows that the effect of the NMT plasma treatment on the action current duration could be mimicked in vitro by 10 p M 3,4DAP: The duration was increased from 1.6 t 0.46

718 Annals of Neurology Vol 38 No 5

November 1775

Table 3. Effect of Pauive Transfer uith Neuromyotonia Plasma on the Superficial Peroneal Nerve of the Mouse'
Duration of the A a P Current (msec) Test Plasma Nerve in Ringer Nerve in Ringer + 500 p M 3,4DAP Control Plasma/ Untreatedb 1.5 12.2

Table 4.Efiect o f Neuromyotonia IgG on Dorsal Root Ganglion (DRG) Cells from the Rat in Tissue Culture"
Test IgG Resting membrane POtentials (mV) Action potentials Threshold (mV) Overshootb (mV) Width' (msec) Repetitive firingd Cell-attached patch Whole-cell patch Spontaneous firingd Cell-attached patch Control I g G

-58 -20 42 4

3 (10)

-52

* 3 (12) *

2.9 13.5

* 0.43 (5)' * 2.8 ( 5 )

0.33 (7) 3.9 ( 7 )

2 2 2

4 (7)
7 (7) l(7)

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

"Mice were treated with plasma from Patient 6 or from blood donors. The width of the current was taken at 10% of its maximum. The number of mice is in parentheses. bResults pooled from 4 mice injected with plasma and 3 untreated mice. 'Different from controls, p = 0.025. 3.4DAP = 3,4-diaminopyridine.

9113'
517'

1/13 3/9g; 21/80h

01 13

21 13

to 3.0 5 0.49 msec (n = 5 , p = 0.010, paired t test). This effect proved rapidly reversible after washout of 3,4DAP. At 500 pM, 3,4DAP caused an approximately tenfold increase in the width of the A a P current (see lower panel of Fig 3 and Table 3). At this concentration of 3,4DAP, the current traces showed a noisy baseline and in some nerves spontaneous individual small action currents could be distinguished. The noise was characterized in a power spectrum which indicated dominant frequencies at 3 and 5 Hz. In a few nerves, when the a fibers were stimulated at a low stimulating voltage in the presence of 500 pM 3,4DAP, repetitive activity could be recognized as individual currents. Their frequency was extremely high (between 500 and 1,000 Hz).

'DRG cells were incubated for 24 hours at 37C in culture medium in the presence of IgG from Patient 6 or controls. The number of cells is in parentheses. bOvershoot of the action potential above 0 mV. 'The width of the action poential was taken at 50% of its maximum. 'Number of cells showing repetitive (spontaneous) firing/total number of cells investigated. eStatisticaUy different from controls ( p = 0.004). 'p = 0.046, test vs control and untreated combined: Fisher's 2 x 2 exact test. Kells treated with control IgG. hCells treated without IgG.

only 2 hours in the presence of 2 mg ml-I of IgG from Patient 6 (not illustrated). Untreated D R G cells, when exposed to 500 pM 3,4DAP, also showed frequent repetitive activity under whole-cell current-clamp conditions (see Fig 4). However, no spontaneous currents were observed in the presence of 3,4DAP. Discussion Evidence far Impaired K+ Channel Function and Involvement of Anti-VGKC Antibodies This report presents converging evidence consistent with the hypothesis that blockade of VGKCs, or a decrease of their number, can be an underlying cause of NMT. The unequivocal evidence of antibodies to VGKC in 2 of the patients, the passive transfer studies in mice, the in vitro effect of purified IgG on D R G cells, and the similarity between the results and those of low doses of the potassium channel blockers 4AP and 3,4DAP strongly support the notion that one form of acquired N M T is autoimmune and that anti-VGKC antibodies play a part. If this proves to be the case, NMT can be added to myasthenia gravis and the Lambert-Eaton myasthenic syndrome as examples of disorders in which ion channels are specifically targeted by autoantibodies. The mechanisms by which the IgG antibodies act are not yet clear; the lack of effect on D R G cells when the incubation was limited to 2 hours suggests that in that situation, a direct block of channel function is not involved.

Effect of IgG on Dorsal Root Ganglion Cells in Tissue Culture Finally, to see whether N M T IgG could affect K+ channel function in vitro, we used tissue culture. Incubation of DRG cells for 24 hours at 37C in the presence of 2 mg ml-I of IgG from Patient 6, compared to incubation in the presence of 2 mg ml-' of control IgG, did not influence the resting membrane potential, the firing threshold, or the size of action potentials and their duration (Table 4). Untreated or control IgG-treated DRG cells showed occasionally repetitive activity when investigated in the cell-attached patch configuration under voltage-clamp mode or whole-cell patch configuration under current-clamp conditions (Table 4, Fig 4). When the cells had been treated with the IgG from Patient 6, repetitive activity was increased from about 10 to 70% (cell-attached mode) or from about 30 to 70% (whole-cell mode). In two cells in the cell-attached patch configuration, spontaneous action currents were observed. Spontaneous and increased repetitive activity was not found when test cells were incubated for

Shillito et al: K f Channels in Neuromyotonia

719

40 20
control IgG

8o I 60 -

NMT IgG

40

>

20 -

I I
I

-20 -40
~

-60

-80

L
0

-80 400
500

100

200

300

100

200

300

400

500

MS

ms

A
6o

>
E

-20

-40

-60

-80

:I--control

6o

3.4OAP

-80 L
0

100

200

300

400

500

100

200 ms

300

400

500

ms

C
Fig 4. Representative trace1 of action potentials of rat dorsal root ganglion cells in tissue culture, measured in the whole-cell patch mode under current clamp, after incubation for 24 hours in the presence of 2 mg ml-' of control or Patient 6 IgG. There was repetitive dctiuity in the neuromyotonia (NMT) IgG-treated (B) but not in a control-treated cell (A). Repetitive action potentials also occurred in the presence (D) but not in the absence (C) of 500 f l 3,4-diaminopyridine (3,4 DAP).

Spec$city of Radioimmunoassc;cy for Anti-VGKC Antibodies

VGKCs derive from alternate splicing of many different VGKC gene products. 3,4DAP and 4AP act on several types, but a-DnTx binds to a limited number of forms only. For example, a-DnTX is ineffective in muscle cells 1271, but it increases ACh release by blocking K f channels of nerve terminals 128) and blocks a fast activating type of K + channel in the nodes of Ranvier of motor nerves 1129-311. The results from the radioimmunoassay with lZSI-a-DnTx-labeled brain VGKCs were above the control range in Patients 2, 3 , and 6, indicating the presence of antibodies to these channels. Results from the remaining 3 patients were not different from control results. The skewing of the NMT results toward values within the control range suggests that the assay lacks the sensitivity to detect antibodies in all patients. Moreover, using a new, i m munohistochemical approach we obtained positive results in a high proportion of NMT patients using recombinant VGKC subtypes (HBK2 and HBK5; I. Hart, C. Waters, A. Vincent, J. Newsom-Davis, unpublished observations, 1995).

Effects at the Neuromusculur Junction Three of the 6 sera from the patients caused an increase of the mean quantal content of end-plates of the mouse diaphragm. These results were consistent with previous evidence that plasma and IgG from Patient 2, injected into mice, increased the efficiency of neuromuscular transmission in vitro, as shown by the increased resistance to the paralysis caused by tubocurarine [12]. The MEPP amplitudes in mice injected with IgG or plasma from NMT Patients 1, 3, and 6 were slightly decreased; this might have been caused by the presence of antibodies to the ACh receptor, since anti-ACh receptor antibodies are sometimes found in NMT patients [9, lo}, but none of these patients were positive for anti-AChR. In the 3 patients with neuropathy (Patients 3-5) some of the clinical symptoms could have arisen as a secondary result of peripheral nerve damage. Nerveinduced continuous muscle fiber activity, which is the hallmark of NMT, could, in principle, be the result of inhibition of acetylcholinesterase (AChE) at the endplate {32}, or due to abnormal or prolonged activity of Na' channels at the nodes of Ranvier. The latter would be difficult to distinguish electrophysiologically from a block of VGKCs. However, the activity of AChE at end-plates of mice treated with Patient 6 plasma was not reduced (P. Molenaar, G. van Kempen, unpublished observations, 1994). In Patient 3, in addition to a low level of anti-VGKC, other autoantibodies including anti-GM and antinuclear antibody were present, consistent with an autoimmune process. How-

720 Annals of Neurology Vol 38 No 5 November 1995

ever, Patients 4 and 5 had no evidence of autoimmunity and the response to plasma exchange in Patient 4 was unclear. In these patients, therefore, the experimental evidence for a role of antibodies is not yet conclusive.
Effects at the Newe It might be expected that the injected human IgG in the mouse had more easy access to the motor nerve terminals than to nodal and paranodal regions in the nerve. However, the fact that action currents in the superficial peroneal nerve were markedly prolonged after treatment with Patient 6 plasma indicates that the blood-nerve barrier was breached to some extent, though neuromyotonia-like symptoms were not seen in the mice. Presumably the proportion of ion channels whose activity was reduced was not sufficient to cause frequent spontaneous or repetitive activity in motor nerves. By contrast, the experiments with the DRG cells showed that when nerve cells were directly exposed to NMT I&, or to 3,4DAP, repetitive activity did indeed occur in vitro. In this connection it is of interest that a-DnTx is a selective blocker of a rapidly activating, sustained K f current of a low conductance K+ channel in DRG neurons {33-351 causing repetitive firing of action potentials. These channels would appear to be a likely target for anti-VGKC antibodies in these cells. At the frog node of Ranvier the number of K + channels is relatively scarce compared to the number of Naf channels; in mammalian nerve the number of K C channels is even smaller [36]. Therefore, it is somewhat surprising that 3,4DAP, and a-DnTx in experiments by others caused repetitive action potentials in the peroneal nerve of the mouse and the DRG cells of the rat. Earlier reports on sciatic nerve from young and adult rabbits indicated that 4AP causes a prolongation of the compound action potential in young rats, but not in adult animals {37-391. The effects of aDnTx and 3,4DAP on the nerve strongly suggest that spontaneous and repetitive movements in NMT are generated in the region of the node of Ranvier through a decrease of the number of available VGKCs. The symptoms of NMT have some resemblance to the spontaneous movements of the Shaker Drosophikz, which carries a mutant gene for a VGKC {40]. Mouse mutants of potassium channel genes that have been described display head waving, body tremors, uncoor4 I]. Recently, episodic dinated gait, and hyperactivity C ataxia, a syndrome in which patients have attacks of generalized ataxia and also experience myokymia due to discharges in peripheral motor nerves, has been associated with point mutations in the potassium channel gene KCNA1 {42]. This hereditary form of NMT appears to involve a change of function rather than a complete loss of one type of K + channel. Our present

findings indicate that autoantibodies to VGKCs are present in some patients with NMT, and could be responsible for their disordered function.
We gratefully acknowledge support by the Medical Research Council and Muscular Dystrophy Group of Great Britain, the Sir Jules Thorn Trust, and the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (to P. C. M.). We thank Dr Anneke Brand for helpful discussions, and Ms Monique van Lint for the purification of IgG.

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