Calcium-Channel Antibodies in the Lambert–Eaton Syndrome and Other Paraneoplastic Syndromes
Vanda A. Lennon, M.D., Ph.D., Thomas J. Kryzer, Guy E. Griesmann, M.S., Padraig E. O'Suilleabhain, M.D., Anthony J. Windebank, M.D., Andreas Woppmann, Ph.D., George P. Miljanich, Ph.D., and Edward H. Lambert, M.D., Ph.D.
Background Voltage-gated calcium channels in small-cell lungcarcinomas may initiate autoimmunity in the paraneoplastic neuromusculardisorder Lambert–Eaton syndrome. The calcium-channel subtypethat is responsible is not known.
Methods We compared the effects of antagonists of L-type, N-type,and P/Q-type neuronal calcium channels on the depolarization-dependentinflux of calcium-45 in cultured carcinoma cells. Serum samplesfrom patients with various disorders were tested for reactivitywith P/Q-type channels solubilized from carcinoma and cerebellarmembranes and N-type channels from cerebral cortex.
Results P/Q-type calcium-channel antagonists were the most potentinhibitors of depolarization-induced 45Ca influx in culturedsmall-cell carcinoma cell lines. Anti–P/Q-type calcium-channelantibodies were found in serum from all 32 patients with theLambert–Eaton syndrome and a diagnosis of cancer and in91 percent of the 33 patients with the Lambert–Eaton syndromewithout cancer. Anti–N-type calcium-channel antibodieswere found in 49 percent of the 65 patients with the Lambert–Eatonsyndrome. Lower titers of anti–P/Q-type and anti–N-typecalcium-channel antibodies were found in 54 percent of 70 patientswith a paraneoplastic encephalomyeloneuropathic complicationof lung, ovarian, or breast carcinoma, 24 percent of 90 patientswith cancer but no evident neurologic complications, 23 percentof 78 patients with sporadic amyotrophic lateral sclerosis,and less than 3 percent of 69 patients with myasthenia gravis,epilepsy, or scleroderma.
Conclusions The high frequency of P/Q-type calcium-channel antibodiesfound in patients with the Lambert–Eaton syndrome impliesthat antibodies of this specificity have a role in the presynapticpathophysiology of this disorder.
Paraneoplastic syndromes are most often associated with small-celllung carcinoma, a relatively common neuroendocrine neoplasm.The autoimmune neurologic syndromes may reflect host immuneresponses against neuron-like components of the tumor cells1(Figure 1). An example is the Lambert–Eaton syndrome,a disorder of neuromuscular transmission caused by antibodiesthat impair the presynaptic release of acetylcholine.10,11
Figure 1. Antibody Assay and Model for the Induction of the Neurologic Spectrum of Autoimmunity.
Small-cell lung carcinomas have antigens in the plasma membrane, nucleus, and cytoplasm that are normally found in neurons of the peripheral and central nervous systems. Examples include the P/Q, N, and L subtypes of voltage-gated calcium channels2,3,4 and neuronal nuclear and cytoplasmic proteins (e.g., HuD,5 NOVA,6 and related RNA-binding proteins). The latter are antigens for paraneoplastic marker antineuronal nuclear autoantibodies type 1 and 2 (ANNA-1 and ANNA-2, also known as anti-Hu and anti-Ri, respectively).1,7,8 The focal necrosis that is characteristic of small-cell carcinomas releases tumor proteins and DNA to cells with antigen-presenting potential.9 Helper T lymphocytes activated in this fashion might therefore initiate the production of autoantibodies against a macromolecular complex of self-proteins. The assay shown detects anti–P/Q-type calcium-channel antibodies in all patients with paraneoplastic Lambert–Eaton syndrome. Antibodies directed at extracellular epitopes may explain the pathogenicity of serum IgG preparations that impair the depolarization-induced release of acetylcholine at neuromuscular synapses when injected into mice.10,11 No causal role is yet ascribed to antibodies against other calcium-channel subtypes. Calcium-channel antibodies complement ANNA-1 and ANNA-2 as markers for encephalomyeloneuropathies that occur with small-cell carcinoma. They also complement ANNA-2 and type 1 anti–Purkinje-cell antibodies as markers for encephalomyeloneuropathies that occur with breast and ovarian carcinomas. The implication, yet to be proved, is of a common immunobiologic basis for all these disorders, which may constitute a spectrum of pathophysiology. Commonly affected regions of the neuraxis are indicated by red dots.
Acetylcholine is released from storage vesicles in the nerveending in response to an action potential. This mechanism requiresthe regulated influx of calcium through voltage-gated channelsin nerve terminals. In the Lambert–Eaton syndrome, thesecalcium channels are the target of pathogenic autoantibodies.10,11Cultured small-cell lung carcinoma cells exhibit voltage-activatedcalcium-channel activity.12 A report that IgG from patientswith the Lambert–Eaton syndrome interferes with this activity13focused attention on subtypes of calcium channels in small-celllung carcinoma.2,3,4,14,15 The subtype of a calcium channeldepends on its 1 subunit (Figure 2), which contains the voltagesensor, antagonist-binding sites, and cation pore. Auxiliarysubunits include 2 and (also for L-type channels of muscleand "95 K" for N-type channels).16,17,18,19 The calcium channelsin small-cell carcinomas were initially reported to be L-like13,14,15and N-like,4,14,15 because of pharmacologic sensitivities todihydropyridines and a snail-derived neurotoxin, -peptide GVIA.A molecular classification of calcium channels, based on DNAsequences of 1 subunits,16 led to the detection of RNA transcriptsfor L, N, and P/Q subtypes of calcium channels (Figure 2) insmall-cell carcinomas.2,3,4 The variety of calcium channelsin tumors might provoke a diversity of autoantibodies that reactwith a particular channel subtype or several subtypes. Antibodiesagainst extracellular segments of calcium channels in neuronscould potentially cause neurologic syndromes.
Figure 2. Classification of Mammalian High-Voltage–Activated Calcium Channels.
The subtype of a calcium channel is determined by its 1 subunit, which is approximately 2000 amino acids long and contains the channel's voltage sensor, antagonist-binding sites, and cation pore. Auxiliary subunits include 2 and (also for L-type channels of muscle and "95 K" for N-type channels).16,17,18,19 The dendrogram was adapted from Zhang et al.17 The degree of homology between any pair of 1 subunits is indicated as the percentage of sequence identity for that pair at a branch point.
The selectivity of -peptides GVIA and MVIIC for the N and P/Q subtypes depends on assay conditions. 125I-labeled MVIIC has a higher affinity for P/Q-type channels than for N-type channels under the conditions used in our study for binding and immunoprecipitation of channels solubilized from human-brain tissue. A rat monoclonal antibody against a variable-region peptide of 1 class B sequence immunoprecipitated 100 percent of GVIA receptors, but less than 0.002 percent of an equivalent amount of solubilized MVIIC receptors (unpublished data). By adding excess unlabeled GVIA peptide to solubilized receptors before introducing 125I-labeled MVIIC, we ensured that the antibodies designated as having specificity for P/Q-type channels in patients with the Lambert–Eaton syndrome were not immunoprecipitating N-type channels that potentially bind 125I-labeled MVIIC.
Class E- or R-type channels are the most recently discovered and least characterized of the high-voltage–activated calcium-channel subtypes. They are resistant to blockade by specific antagonists of the other types of calcium channels. Their physiologic role remains obscure. Dihydropyridines are calcium-channel blockers, such as nifedipine and nicardipine, used to treat hypertension. Data were obtained from Sher et al.,15 Oguro-Okano et al.,2,3 Codignola et al.,4 Birnbaumer et al.,16 Olivera et al.,18 Uchitel et al.,20 Sugiura et al.,21 Hillyard et al.,22 and Kristipati et al.23
Toxins from various venoms are useful for analyzing the diversecalcium channels that initiate the release of neurotransmitters.18,20,21The P/Q subtype is probably the predominant mediator of neuromusculartransmission. In mice, evoked acetylcholine release is blockedat the P/Q type of synapse by a cone-snail -peptide called MviiC21and a funnel-web–spider polyamine called FTx.20 Antagonistsof L-type and N-type channels have no effect.
We report evidence that small-cell lung carcinoma cell linesfrom patients with or without neurologic autoimmunity have high-affinityreceptors for MviiC, a P/Q-type calcium-channel antagonist.18,21,22,23,24Almost all the patients with the Lambert–Eaton syndrome,whether or not they had evidence of cancer, had serum antibodiesto these high-affinity receptors for MviiC. About half the patientsalso had antibodies to receptors for the N-type calcium-channelantagonist GVIA. Antibodies against calcium channels were alsofound, at lower frequencies and titers, in patients with paraneoplasticencephalomyeloneuropathies associated with lung, ovarian, andbreast cancers; in a minority of patients with cancer withoutevident neurologic dysfunction; and in patients with sporadicamyotrophic lateral sclerosis.
Methods
Small-Cell Lung Carcinoma Cell Lines
The small-cell lung carcinoma cell lines SCC-9 and SCC-15 wereestablished in the neuroimmunology laboratory of the Mayo Clinicand have been characterized in earlier studies.14,25,26,27 Cellswere grown in RPMI-1640 medium supplemented with 10 percentcalf serum.
Calcium-45 Influx Assays
The depolarization-dependent influx of 45Ca, assayed as describedelsewhere,26 was determined for each specified condition byexposing tumor cells to 90 mM potassium chloride for one minuteat 37°C. Base-line 45Ca influx in 4.7 mM potassium chloridewas subtracted from influx in 90 mM potassium chloride.
Calcium-Channel Antagonists
The -peptide MviiC was synthesized, labeled with iodine-125,and characterized as described previously22; -peptides GVIAand Aga-IVA were obtained from Peninsula Laboratories (Belmont,Calif.) and the Peptide Institute (Osaka, Japan). Nifedipine,a dihydropyridine antagonist of L-type calcium channels, wasobtained from Sigma (St. Louis).
Serum Samples
The study protocol was reviewed and approved by the institutionalreview board at the Mayo Clinic. Patients gave oral consentfor their serum to be used in studies of antineuronal antibodies.Serum was obtained from patients with specified neurologic orautoimmune disorders or cancer and from equal numbers of consecutivenormal subjects, some of whom had a history of tobacco use,and stored at -20°C.
Preparation and Assays of Receptors for -Peptides MviiC and GVIA
Membranes from homogenized small-cell lung carcinoma cell lines25and human cerebellar28 and cerebral cortical29 tissues obtainedat autopsy were solubilized for two hours at 4°C in buffercontaining digitonin (4.5 percent), HEPES (50 mM), glycerol(20 percent), aprotinin (1 kallikrein inhibitory unit per milliliter),pepstatin A (0.1 µg per milliliter), and phenylmethylsulfonylfluoride (2 mM), pH 7.5. The formation of complexes with radioligand,quantitation by glass-fiber filtration, and immunoprecipitationhave been described elsewhere.30 Receptors that formed complexeswith 125I-labeled MviiC or 125I-labeled GVIA (20 pM and 150pM, respectively) were incubated for 16 hours at 4°C withpatients' serum samples (5 µl, or 10-fold dilutions) induplicate in a final volume of 320 µl, before goat antiserumagainst human immune globulins was added for immunoprecipitation.
Results
Pharmacologic Profile of Calcium-Channel Antagonists
As reported previously,13,14,26 brief exposure to high concentrationsof potassium chloride stimulates the influx of 45Ca into culturedsmall-cell carcinoma cell lines. Figure 3A and Figure 3B showsthe pharmacologic sensitivity of depolarization-dependent 45Cainflux for SCC-9, a prototypic small-cell lung carcinoma cellline, and SCC-15, a cell line from a patient with the Lambert–Eatonsyndrome. The -peptide MviiC, at micromolar concentrations,reduced potassium chloride–evoked calcium influx by 60to 90 percent. Micromolar concentrations of another -peptide,Aga-ivA, reduced the depolarization-dependent influx of 45Caby 60 percent in these cell lines. GviA, an -peptide antagonistof N-type channels, reduced stimulated 45Ca influx minimally(20 to 40 percent at 100 µM), contrary to earlier reportsthat it had a potent inhibitory effect on small-cell carcinomacalcium channels.4,14 Nifedipine reduced stimulated 45Ca influxonly at millimolar concentrations, well above the submicromolarconcentrations that specifically block L-type channels.31 Theseresults suggest that calcium channels with P/Q-type propertiesaccount for most of the depolarization-dependent influx of calciumin small-cell carcinoma cell lines, regardless of whether thepatient from whom the cells originated had a neurologic syndrome.
Figure 3. Pharmacologic Sensitivity of Depolarization-Dependent 45Ca Influx in Two Lines of Small-Cell Lung Carcinoma Cells.
SCC-9 (Panel A) is from a 60-year-old man without paraneoplastic neurologic complications; SCC-15 (Panel B) is from a 59-year-old woman with the Lambert–Eaton syndrome and serum antibodies reactive with neuronal receptors for the -peptides MVIIC (titer, 49 pM) and GVIA (titer, 133 pM). Each point represents the mean value of three to seven experiments done in duplicate, expressed as the percentage of potassium-stimulated 45Ca influx (i.e., influx at 90 mM potassium chloride minus influx at 4.7 mM potassium chloride) in the absence of drug. DMSO denotes dimethylsulfoxide, which was used as a vehicle for nifedipine.
Quantitation and Immunoprecipitation of -Peptide Receptors
To optimize our assay for detecting antibodies against P/Q-typecalcium channels,28,32 we first tested solubilized membranesfrom small-cell lung carcinomas and human cerebellum for receptorsfor MviiC using a filtration assay to quantitate binding sitesfor 125I-labeled MviiC. SCC-9 membranes yielded approximately2 fmol of receptors per milligram of protein, and cerebellarmembranes approximately 1300 fmol per milligram. The apparentdissociation constant (Figure 4) (the MviiC concentration requiredto saturate 50 percent of specific receptors) was 75 pM fordigitonin-solubilized human cerebellar membranes, a value consistentwith the dissociation constant for intact rat and bovine cerebellarmembranes.23 A serum reactive with P/Q-type calcium channels(from a patient with the Lambert–Eaton syndrome, identifiedin our preliminary studies28,32) immunoprecipitated receptorsfrom both small-cell lung carcinoma and cerebellum. Despitean approximately 50 times greater preference for P/Q-type calciumchannels,23 MviiC can bind to both P/Q-type and N-type channels.At the concentrations of radiotracer used in our experiments(approximating the dissociation constant for P/Q channels),virtually all 125I-labeled MviiC binding was predicted to beto P/Q-type channels.23 To determine whether N-type calciumchannels with a low affinity for 125I-labeled MviiC were detectedin the immunoprecipitation assay, we retested in duplicate serumsamples from 56 patients with the Lambert–Eaton syndromethat immunoprecipitated 125I-labeled MviiC receptor complexes,using a receptor preparation containing a 1400-fold excess ofunlabeled GviA to render N-type channels inaccessible for radiolabelingby 125I-labeled MviiC. Immunoprecipitation of 125I-labeled MviiC–receptorcomplexes was not significantly reduced (the mean [±SE]value obtained with pretreatment was 91±1 percent ofthe mean value obtained without pretreatment). This indicatedspecific immunoprecipitation of P/Q-type calcium channels.
Figure 4. Specific Binding of 125I-labeled MVIIC to Digitonin-Solubilized Membranes Prepared from Cerebellar Cortex Obtained at Autopsy from a Patient without Neurologic Disease.
The binding was determined by filtration with glass-fiber filters; 8.7 µg of protein was incubated with 125I-labeled MVIIC at 4°C for 5.5 hours. The amount of nonspecific binding in the presence of 1.73 µM unlabeled MVIIC was subtracted from the total.
Frequency of Anti–P/Q-Type and Anti–N-Type Calcium-Channel Antibodies
We used cerebellum as the source of P/Q-type channels to screenserum samples from the patients (Figure 5 and Table 1) becauseof its high density of MviiC receptors. N-type channels (GviAreceptors) were obtained from human cerebral cortical membranes.29The serum samples of all 32 patients who had the Lambert–Eatonsyndrome and cancer were positive for anti–P/Q-type channelantibodies. Among 33 patients with the syndrome but withoutcancer, 30 (91 percent) had anti–P/Q-type channel antibodies.This high frequency suggests that autoantibodies with specificityfor P/Q-type calcium channels have a pathogenic role in thesyndrome. Only 1 of 47 normal subjects (2 percent) and 1 of69 patients (1 percent) with myasthenia gravis, epilepsy, orscleroderma had anti–P/Q-type calcium-channel antibodies.
Figure 5. Distribution of Values for P/Q-type Calcium-Channel Antibodies in Serum Samples from Patients with Various Types of Cancer and Neurologic Disorders and from Normal Subjects.
The horizontal line at 20 pM represents the upper limit of the normal range (2 SD above the mean value for 47 normal subjects). Patients with cancer and the Lambert–Eaton syndrome had primary lung carcinoma (•) or some other cancer (); patients with paraneoplastic encephalomyeloneuropathies had small-cell lung carcinoma (or ANNA-1 or ANNA-2 autoantibody as a marker of small-cell lung carcinoma1) (•) or ovarian or breast carcinoma (or PCA-1 or ANNA-2 as a corresponding marker autoantibody1) (); patients with cancer and no evident neurologic dysfunction had small-cell lung carcinoma (•) or ovarian carcinoma (). Patients with no evidence of cancer had the Lambert–Eaton syndrome, amyotrophic lateral sclerosis, or myasthenia gravis (•) or idiopathic epilepsy (), or they were normal subjects matched to patients with the Lambert–Eaton syndrome for age, sex, and smoking habits. Results for 28 patients with scleroderma were all negative and are not shown.
Table 1. Frequency of Antibodies to P/Q-Type and N-Type Voltage-Gated Calcium Channels in Patients with Various Neurologic, Neoplastic, and Autoimmune Disorders and in Normal Subjects.
In results consistent with our earlier studies using N-typechannels (solubilized from small-cell carcinomas with the detergent3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate [CHAPS]30and from cerebral cortical membranes with digitonin19,29), wefound antibodies against N-type calcium channels in 19 of 26patients (73 percent) with the Lambert–Eaton syndromeand primary lung carcinoma (small cell, squamous cell,30 oradenocarcinoma) (Table 1). These antibodies were significantlyless common among patients with the Lambert–Eaton syndrome(12 of 33, or 36 percent) who had no evidence of cancer or whohad cancers other than lung cancer (1 of 6, or 17 percent; P<0.01by chi-square analysis).
Serum from patients with cancer and no evident neurologic dysfunctionhad a relatively low frequency of antibodies against P/Q-typeand N-type calcium channels (20 of 71 patients with small-celllung carcinoma, or 28 percent; and 2 of 19 with ovarian carcinoma,or 11 percent) (Figure 5 and Table 1). Median values were relativelylow in this group (P/Q-type, 43 pM and 72 pM, respectively;and N-type, 67 pM and 52 pM, respectively). Of 70 patients whohad small-cell lung or ovarian or breast carcinoma with a paraneoplasticencephalomyeloneuropathic complication but without evidenceof the Lambert–Eaton syndrome, 38 (54 percent) had anti–calcium-channelantibodies (19 P/Q-type only, 10 N-type only, and 9 both types).Median values (P/Q-type, 83 pM; and N-type, 179 pM) were betweenthose of patients with the Lambert–Eaton syndrome andthose of patients with cancer and no evident neurologic dysfunction.Anti–calcium-channel antibodies were also found in 18of 78 patients (23 percent) with amyotrophic lateral sclerosis(Table 1). These findings suggest that antigenically relatedcalcium channels of the P/Q, N, or L type may be targets forpathogenic autoantibodies in disorders other than the Lambert–Eatonsyndrome.
Discussion
Our finding of anti–P/Q-type calcium-channel autoantibodiesin 95 percent of 65 patients with the Lambert–Eaton syndromeimplies that these antibodies are a determinant of presynapticpathophysiology in this disorder. The low frequency of theseantibodies (2 percent) in patients with myasthenia gravis orscleroderma and in normal subjects supports the clinical usefulnessof detecting antibodies with brain-derived antigens that havehigh-affinity receptors for -peptides.
Different calcium-channel 1 subunits have regions of considerableamino acid sequence homology (Figure 2), which presumably representhomologies of structure and antigenicity. About half the patientswith the Lambert–Eaton syndrome had antibodies reactivewith N-type calcium channels. This finding may reflect immunityagainst homologous regions of 1 subunits of N-type and P/Q-typecalcium channels. However, in some serum samples we found evidenceof independent responses to N-type and P/Q-type calcium channels.In 15 patients, antibodies with specificity for N-type calciumchannels were present in higher titers than antibodies of P/Q-typespecificity. No pathophysiologic role has been ascribed to antibodieswith specificity for N-type calcium channels. Various frequencieshave been reported in patients with the Lambert–Eatonsyndrome.29,30,34,35 The findings in the present study confirmour earlier finding that the detection of N-type calcium-channelantibodies in a patient with the Lambert–Eaton syndromeincreases the likelihood of discovering an underlying primarylung cancer. In contrast, nearly all patients with the Lambert–Eatonsyndrome, with or without a tumor, are seropositive for anti–P/Q-typecalcium-channel antibodies.
Immunoprecipitation assays of the type used in this study candetect autoantibodies with and without pathogenic potential.19In living neurons, most of the structure of the calcium-channelprotein is inaccessible to antibodies because it is largelysequestered in the plasma membrane and cytoplasm (Figure 1).Solubilization with detergent, however, would render more antigenicsites accessible in vitro. Using Western blotting, Rosenfeldet al.36 found that three of seven patients with the Lambert–Eatonsyndrome had antibodies to human-brain calcium-channel subunits,which are predicted to be cytoplasmic.19 Leveque et al.37 suggestedthat the primary target of Lambert–Eaton antibodies mightbe the neurotransmitter-vesicle protein synaptotagmin ratherthan the calcium-channel protein itself. Synaptotagmin is exposedin the synapse transiently during exocytosis of transmitter,and in certain detergents it associates noncovalently with calcium-channelproteins. In agreement with the observations of Leveque et al.on rat-brain GviA receptors extracted in Triton X-100,37 wefound that human-brain GviA receptors (and MviiC receptors)in CHAPS detergent were immunoprecipitated by a mouse antisynaptotagminmonoclonal antibody (MAb 48, provided by Dr. Louis Reichardt,University of California, San Francisco). However, in digitonin,the detergent that we have found optimal for the immunoprecipitationof calcium channels by IgG from patients with the Lambert–Eatonsyndrome,28,32 neither GviA nor MviiC receptors were appreciablyimmunoprecipitated by antisynaptotagmin antibody.
Small-cell lung carcinoma and other cancers are sometimes complicatedby paraneoplastic neurologic disorders other than the myasthenicsyndrome (Figure 1). We studied 70 patients with an assortmentof these other paraneoplastic neurologic disorders. Most patientshad a marker antibody in their serum appropriate to their tumor(Table 1). Patients with encephalopathy, cerebellar ataxia,myelopathy, or peripheral neuropathy had type 1 or type 2 antineuronalnuclear autoantibodies. These serologic markers of small-cellcarcinoma1 are directed at nuclear antigens in neurons and tumors.1,5,6,7Female patients with breast carcinoma who had midbrain encephalitisand myelopathy also had type 2 antineuronal nuclear autoantibodies,1,6,8and female patients with ovarian or breast carcinoma who hadsubacute cerebellar ataxia had type 1 anti–Purkinje-cellcytoplasmic antibodies.1,33 In 38 of these patients (54 percent),we detected anti–P/Q-type or anti–N-type calcium-channelantibodies.
It has not yet been shown that antibodies against a particularsubtype or extracellular segment of calcium channel can impairneuromuscular transmission. Nor is it known whether any antibody(or T cells) against calcium channels (Figure 1) can actuallycause a paraneoplastic encephalomyeloneuropathy. Evidence mightbe obtained experimentally by injecting rats or mice3 with antibodiesof a defined calcium-channel specificity (affinity-purifiedor monoclonal) or by immunizing animals with antigenic polypeptidesof 1-subunit sequences. The different neurologic presentationsof seropositive patients in the three groups with cancer (theLambert–Eaton syndrome, encephalomyeloneuropathy, andapparent neurologic normality) (Figure 5) might be explainedby polyclonality of an individual patient's immune responsesand by a multiplicity of epitopes immunogenic for B cells inany given calcium-channel subtype. An example is a patient westudied who had cerebellar ataxia related to ovarian carcinomabut no detectable neuromuscular transmission defect.38 She hadserum antibodies reactive with N-type calcium channels (titer,322 pM), but negligible P/Q-type antibodies (titer, 16 pM).In contrast is a patient who had clinical and electrophysiologicsigns of the Lambert–Eaton syndrome without evident centralnervous system problems or cancer; this patient had serum antibodiesreactive with P/Q-type calcium channels at 918 pM, which was16 times higher than the level of N-type calcium-channel antibodies(57 pM). In mice injected with the IgG fraction of serum fromthat patient,11 a profound neuromuscular transmission defectdeveloped.
Smith et al.39 reported that 75 percent of patients with sporadicamyotrophic lateral sclerosis and 75 percent of patients withthe Lambert–Eaton syndrome have antibodies that bind toL-type calcium channels purified from skeletal muscle. Muscleis not primarily involved in the pathophysiology of either disorder.Llinas et al.40 reported that IgG prepared from the serum ofa patient with amyotrophic lateral sclerosis affected P-typecalcium-channel activity in dissociated cerebellar Purkinjecells. We found P/Q-type and N-type calcium-channel antibodiesin a minority of the 78 patients with amyotrophic lateral sclerosiswe studied. This result may also reflect the detection of antibodiesthat cross-react with multiple calcium-channel subtypes (Figure 2).
These observations provide evidence of the antigenic relatednessof different classes of high-voltage–activated calciumchannels. Although antibodies against cytoplasmic epitopes arenot anticipated to be pathogenic, they might serve as markersof either antitumor immunity or a neurologic spectrum of autoimmunity(Figure 1). On the other hand, as illustrated by the Lambert–Eatonsyndrome, antibodies specific for calcium-channel epitopes thatare exposed in discrete anatomical regions may have a pathogenicrole in both paraneoplastic and nonparaneoplastic neurologicdisorders.
Supported in part by a grant (to Dr. Lennon) from the NationalCancer Institute (CA-37343).
We are indebted to Jean Erickson, Lisa Baker, Mary Lohse, NormanPinsky, Evelyn Posthumus, and Jim Thoreson for excellent technicalassistance, and to Laszlo Nadasdi for providing -conopeptideMviiC.
Source Information
From the Departments of Immunology, Neurology, and Laboratory Medicine–Pathology (V.A.L., T.J.K., G.E.G., P.E.O., A.J.W., E.H.L.), Mayo Clinic, Rochester, Minn., and the Neurex Corporation (A.W., G.P.M.), Menlo Park, Calif. Preliminary reports of this work were presented at the Annual Meeting of the Society for Neuroscience, Washington, D.C., November 7–12, 1993, and the Eighth International Congress on Neuromuscular Diseases, Kyoto, Japan, July 10–15, 1994.
Address reprint requests to Dr. Lennon at the Neuroimmunology Laboratory, Rm. 828, Guggenheim Bldg., Mayo Clinic, Rochester, MN 55905.
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1 subunit (Figure 2), which contains the voltage sensor, antagonist-binding sites, and cation pore. Auxiliary subunits include 
and 
(also 
for L-type channels of muscle and "95 K" for N-type channels).16,17,18,19 The calcium channels in small-cell carcinomas were initially reported to be L-like13,14,15 and N-like,4,14,15 because of pharmacologic sensitivities to dihydropyridines and a snail-derived neurotoxin, 
-peptide GVIA. A molecular classification of calcium channels, based on DNA sequences of 
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); patients with paraneoplastic encephalomyeloneuropathies had small-cell lung carcinoma (or ANNA-1 or ANNA-2 autoantibody as a marker of small-cell lung carcinoma