LAMBERT-EATON SYNDROME ANTIBODIES TARGET MULTIPLE SUBUNITS OF VOLTAGE-GATED Ca2+ CHANNELS
Ravindra K. Hajela, Ph.D., Kristin M. Huntoon, D.O, Ph.D., and William D. Atchison, Ph.D.
Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan, USA.
Address all correspondence including reprint requests to: Dr. William D. Atchison Department of Pharmacology and Toxicology Michigan State University 1355 Bogue Street B331 Life Sciences Building East Lansing, MI 48824-1317 phone: (517) 353-4947 fax: (517) 432-1341 e-mail: [email protected]
Running Title: LEMS IgG and Cav channel subunits Dr. Huntoon’s Current Address: Surgical Neurology, National Institute of Neurological Disorders and Stroke, National Institute of Health, Bethesda, Maryland
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/mus.24295
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LEMS IgG and Cav channel subunits, Page 2 Abstract Introduction: Lambert Eaton Myasthenic syndrome (LEMS) is an autoimmune presynaptic neuromuscular disorder. Autoantibodies against subunits of voltage gated calcium channels (VGCCs) associated with acetylcholine release are thought to cause LEMS. Methods: HEK293 cells expressing specific individual recombinant subunits of α1A, B, C and E, β3, and α2δ of human neuronal VGCCs were exposed to antibodies from 3 LEMS patients, a patient with small cell lung carcinoma, and 1 with myasthenia gravis. Results: All LEMS patient antibodies bound to cells containing any of the α1 or β3 alone or combined with α2δ subunits, but not α2δ alone. Autoantibodies from the patient with small cell lung carcinoma but not the myasthenia gravis patient targeted the same VGCC subunits. Conclusion: autoantibodies from LEMS patients bind directly to multiple VGCC α1 subunits as well as the β3 subunit. Thus, multiple components of the presynaptic VGCC complex are prospective targets for antibodies in LEMS.
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LEMS IgG and Cav channel subunits, Page 3 Keywords: 1. Lambert-Eaton Myasthenic Syndrome 2. Voltage-gated Ca2+ Channels 3. Neuromuscular Disease 4. Small Cell Lung Carcinoma 5. Acetylcholine Release
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LEMS IgG and Cav channel subunits, Page 4 Introduction Lambert-Eaton Myasthenic Syndrome (LEMS) is a paraneoplastic, autoimmune disorder of neuromuscular transmission. It results in skeletal muscle weakness which involves the lower more than upper extremities1. Muscle weakness is due to a reduction in the number of quanta of acetylcholine (ACh) released by the motor nerve terminal in response to a nerve impulse.2 Clinical manifestations of the disease are ascribed to disruption of Ca2+ influx through voltagegated Ca2+ channels (VGCCs) in the motor nerve terminal, a process essential for release of ACh.3 Although there are various subtypes of VGCCs, the P/Q-type (Cav2.1) are involved primarily in ACh release from adult mammalian motor nerve terminals.4 Other Ca2+ channel phenotypes, such as N-type (Cav2.2) and L-type (Cav1.3), are responsible for transmitter release from peripheral autonomic nerve and central terminals or hormone release respectively,5-10 and some are also affected in LEMS10. Approximately two-thirds of LEMS patients also have small cell lung carcinoma (SCLC) in which the cancer cells overexpress several types of VGCCs.11 LEMS is thought to be triggered by cross-reactive binding of circulating autoantibodies to motor nerve terminals, presumably produced initially in response to the VGCC complex components. Conceptually, autoantibodies against any of the several proteins that form a functional VGCC complex, or proteins that present these antigens to the immune system, can be generated and found in patient sera. Indeed evidence for all of these possibilities has been presented. For example, approximately 64% of paraneoplastic LEMS patients with SCLC also have antibodies against SOX1, a transcription factor protein strongly expressed in the developing nervous system, suggesting the possibility of a very early marker of the future predisposition to LEMS/SCLC.12 All the present evidence suggests that antibodies against proteins closely linked to the VGCC
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LEMS IgG and Cav channel subunits, Page 5 functional complex could be found in some patients and not others.13 Therefore, the circulating autoantibodies against components of VGCCs are prime candidates for disrupting motor nerve terminal function.14-18,2 However, whether antibodies are generated against more than 1 specific subtype of VGCC or multiple subunits of those channels is not yet known with certainty. In vitro studies of Ca2+ influx during depolarization19 or measurement of inward currents through VGCCs using divalent charge carriers show reduction when cells in culture (including those of neuronal origin or small cell lung carcinoma) are exposed to LEMS antibodies.18-20 Perineurial-generated Ca2+ currents from nerve terminals of mice treated chronically with LEMS patient sera or plasma are reduced in amplitude in general but show an apparent upregulation of L-type current and dihydropyridine sensitivity of neuromuscular transmission.21,22 Both low voltage-activated (LVA) and high voltage-activated (HVA) Ca2+ currents were reduced in amplitude in dorsal root ganglia from mice treated with LEMS patient sera, but currents were reduced preferentially in motor neurons compared to sensory neurons in culture, and L-type currents were spared.23,24 However, in HEK293 cells stably expressing Cav2.1 (P/Q-type), Cav2.2 (N-type), Cav1.2 (L-type), and Cav2.3 (R-type) VGCCs, LEMS patient IgG caused a significant reduction in K+-stimulated increase of Ca2+i only in Cav2.1 (P/Q-type)-expressing cells.25,26 The intracellular β subunits have also been implicated in the etiology of LEMS, because a screen of an expression cDNA library using LEMS sera resulted in isolation of a β subunit cDNA clone.27 Recognition of the protein in western blots produced expression of the cDNA clone of the β subunit.28 Results of prior pharmacological studies, toxin binding, current recording, or other indirect studies29,30,25,26, taken together, demonstrate that 1 or more VGCC subunits can be targeted by LEMS antibodies. However, no cytological evidence using antibodies from LEMS
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LEMS IgG and Cav channel subunits, Page 6 patient sera or plasma has been presented. In this study, we provide direct immunocytological evidence that multiple VGCC subunits can bind circulating autoantibodies from LEMS patients and an SCLC patient, but not a myasthenia gravis (MG) patient. In order to determine which individual subtypes or subunits of VGCCs are recognized by the circulating autoantibodies in the LEMS patient sera and avoid confounding effects associated with the presence of the secretory complex or other potential targets, we used a heterologous system transiently expressing cDNA clones of specific subunits of human neuronal HVA VGCCs in HEK293 cells in culture.
Materials, Methods and Experimental Design HEK293 cells #CRL-1573 were purchased from American Type Culture Collection (Rockville, MD). Expression cDNA clones for human neuronal VGCC α1A-2 (from cerebellum31), α1B-1 (from IMR32 cells of human neuronal origin),32 α1C-1 (from hippocampus, personal communication, Dr. Mark Williams, Merck Research Laboratories) or α1E-3 (from hippocampus33), and α2bδ (from brain stem and basal ganglia34) and β3-a (from hippocampus, personal communications, Dr. Mark Williams, Merck Research Laboratories) subunits were provided by Dr. Kenneth Stauderman of SIBIA Neurosciences, now Merck Research Laboratories, San Diego, CA. The LEMS, SCLC, and MG patient sera for IgG preparation were provided by Drs Andrew Massey, University of Kentucky Medical Center, Lexington, KY, Eva Feldman and James Albers, University of Michigan Medical Center, Ann Arbor, MI, and Shin Oh, University of Alabama Medical Center, Birmingham, AL. Control human plasma was obtained as outdated donated material from the blood bank of the American Red Cross, Lansing, MI, courtesy of Dr. Donald Penner. All sera/plasma were supplied to us with patient age and gender as the only identifiers. Samples of LEMS, SCLC, and MG plasma were collected during
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LEMS IgG and Cav channel subunits, Page 7 the course of routine plasma exchange therapy with normal informed consent of the patients in accordance with the approval of regulatory Human Subject Committees of the respective institutions. All reagents were off the shelf pure or ultra pure laboratory grade unless specifically noted. HEK293 cells were grown at 37oC in Eagle Modified Essential Medium fortified with 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 10% (w/v) fetal bovine serum, and penicillin, streptomycin, antimycotic, and antibiotics (GIBCO-BRL/Invitrogen Technologies, Gaithersburg, MD) in a 5% CO2 environment. One day before gene transfer, cells were plated at a density of 5x105 on poly Llysine-coated 25 mm round cover slips in 35 mm culture dishes. Cells were transfected with a mixture of plasmids containing expression cDNA clones of human neuronal VGCC subunits α1A2,
α1B-1, α1C-1, or α1E-3, either individually or together with α2bδ and β3-a and a green fluorescent
protein (GFP) cDNA clone, using Fugene 6 (Roche Molecular Biochemicals, Indianapolis, IN) and following the manufacturer’s instructions. Reactions contained a total of 3 µl of Fugene 6 and 1 µg plasmid DNA containing the 3 VGCC subunits in 1:1:1 molar ratio, or only the specific subunit. GFP plasmid was also included at 20% of the total DNA. Two to 3 days were allowed for optimal transient expression of proteins, at which time the cells were examined for GFP expression using an Olympus IX70 fluorescence microscope (Olympus Optical, Tokyo, Japan) with a mercury arc burner at excitation wavelength of 460-490 nm and emission wavelength of 510-550 nm for GFP expression and excitation wavelength of 510-550 nm and emission wavelength of 580-590 nm for rhodamine fluorescence. LEMS patient IgG samples were purified using affinity column chromatography and antihuman IgG sepharose or agarose beads (Sigma-Aldrich, St. Louis, MO). Protein content was
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LEMS IgG and Cav channel subunits, Page 8 quantitated using the Bio-Rad DC protein assay kit (Bio Rad, Hercules, CA) with bovine serum albumin as a standard. For immunocytochemistry, each cover slip was rinsed 3 times (3 min/rinse) with Dulbecco phosphate buffered saline (DPBS) in (mM): CaCl2 45, KCl 1.34, KH2PO4 0.74, MgCl2 0.25, NaCl 70, NaH2PO4 4, (pH at 7.4). Cells were fixed with 4% (w/v) paraformaldehyde in DPBS (2 ml/cover slip) for 10 min at room temperature of 23-25oC followed by another set of rinses as above. Fixed cells were permeabilized by soaking the cover slips in 0.2% (w/v) Triton X-100 in DPBS for 10 min at room temperature. Following a set of 3 rinses as above, the cover slips were incubated for 10 min at room temperature in a blocking solution. In experiments using patient IgG, the blocking solution contained 7.8% (w/v) goat serum, 5.5% (w/v) horse serum, and 35.25 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] in DPBS. For experiments using anti-subunit antibodies, blocking solution contained 3% (w/v) bovine serum albumin (Sigma-Aldrich, St. Louis, MO) in DPBS. After blocking, the cover slips were incubated in 1.2 ng/ml of LEMS/MG/SCC IgG or anti-subunit antibodies (Alomone Labs Ltd., Jerusalem, Israel) made up in blocking solution and turned inverted on cavity slides (Fisher Scientific, Itasca, IL). These slides were incubated in a dark, humidified chamber for 1 hr at room temperature, then for 30 hrs at 4oC. Cells were subsequently rinsed 3 times as before, followed by incubation in 1:5000 dilution of rhodamine red-X-conjugated donkey anti-human IgG (Jackson Immuno Research Laboratories Inc., West Grove, PA) or rhodamine-conjugated donkey anti-rabbit IgG antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), respectively. Cover slips were gently rinsed again 3 times with blocking solution before examination under the fluorescence microscope. No attempt was made to quantitate the epifluorescence. Two sets of experiments were performed. In the first, fully functional VGCCs of
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LEMS IgG and Cav channel subunits, Page 9 predetermined subtype were expressed in HEK293 cells using an equimolar combination of a specific α1 subunit with constant α2δ and β subunits. Functional expression of the specific subtype has previously been demonstrated using whole cell recordings.35 The second set of experiments was designed to determine which subunit(s) were specifically recognized by the LEMS antibodies. cDNA of only 1 subunit was used in any given transfection. In all experiments, the cells were fixed and permeabilized before performing the immunocytological screening. Permeabilization was done to facilitate antibody accessibility to target VGCC subunits in the cytoplasm. Each experiment included various sets of positive and negative controls. To conserve space, most of these results are not shown in the manuscript. One set of cover slips with specific subunit-transfected cells was treated with commercially available anti-subunit-specific primary antibodies generated in rabbits in order to verify the protein expression and to establish binding of antibody. These cover slips were treated further with rhodamine-conjugated donkey antirabbit secondary antibody to visualize the binding of the primary antibody. Cover slips treated with either only anti-subunit specific primary antibody, with only rhodamine-conjugated donkey anti-rabbit secondary antibody, or only rabbit serum and the labeled secondary antibody never showed any fluorescence (Results not shown). This assured us that any signal visualized and recorded was due to binding of the labeled secondary antibodies to the anti-subunit specific antibody bound to the VGCCs expressed in the transfected cells growing on the cover slips. Specificity of anti-subunit antibodies was tested by treating cells with each of the anti-subunit antibodies separately as well as treating the cells with antibodies for subunits other than those with which the cells were transfected. No cross-reactivity was ever seen among subunit subtypespecific antibodies. (Results not shown.) Cells grown on poly L-lysine-treated test cover slips
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LEMS IgG and Cav channel subunits, Page 10 were treated with LEMS patient IgG followed by rhodamine-conjugated donkey anti-human secondary antibodies. Negative controls for this set of experiments included incubation with only LEMS patient IgG, only the labeled secondary antibodies, and control human serum plus secondary antibodies. No fluorescence was seen in any of the controls (Results not shown.). Finally, untransfected cells were treated with patient IgG or anti-subunit antibody and respective labeled secondary antibody; they exhibited no binding or autofluorescence.
Results Figures 1-4 depict epifluorescence generated in HEK cells either transfected with single specific subunits of HVA VGCCs, whole channel complexes, or controls. In each figure, the left panel depicts GFP fluorescence, and the same optical field in the right panel depicts fluorescence due to binding of specific secondary antibody. Often, greater numbers of cells are seen as antibody positive than as GFP positive. This is due to the fact that only 20% of transfecting DNA used was for GFP, giving the statistical probability of 4 non-green cells being transfected with VGCC subunit DNA for every cell transfected with GFP. The different pattern of fluorescence in different cells is due to the distribution of the target proteins variously in the cytoplasm and the membrane. The punctate signal on the perimeter represents the membrane-bound signal, while the general diffuse signal is from the top, the bottom, and the cytoplasm of the cell. The apparent opacity is the very dense nuclear area. Subunits not yet fully processed would be found in the cytoplasm. Cytoplasmic localization would also occur when only a single subunit was transfected, because the transport and proper insertion of the channel in the cell membrane is highly dependent on accessory subunits, especially the β subunit. Figure 1 demonstrates the binding of subunit-specific antibodies to cells transfected with
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LEMS IgG and Cav channel subunits, Page 11 specific subunits to serve as a positive control of expression of transfected cDNAs. Also included in the control experiments were assays to check for nonspecific binding of either the wrong antibody or only the secondary antibody to transfected cells and LEMS patient IgG and secondary antibody to nontransfected cells. Figure 2 shows the binding of IgG from 1 representative LEMS patient to cells transfected with the full complement of cDNA for all 3 subunits of VGCCs including 1 of the subunits α1A-2, α1B-1, α1C-1, or α1E-3 together with α2bδ and β3-a. Affinity-purified IgG from all 3 LEMS patients showed binding to cells transfected with each of the 4 sub-types of VGCCs. (Results only shown for a representative patient.) These results provide direct evidence that LEMS patient autoantibodies are directed against multiple subtypes or multiple subunits of VGCCs. It has been shown previously that LEMS patient IgGs immunoprecipitate more than 1 type of VGCC from several kinds of human and rat cells. However, Pinto et al., 26 and Sher et al.,3 reported that functions of only P/Q-type VGCCs are impaired in HEK293 cells stably transfected with expression cDNA clones. These contrasting observations, although obtained using different techniques, may be due to differences in sensitivity of techniques or use of a common α2δ and β3 subunit with different α1 subunits in heterologous expression experiments such as ours and a different complement of these accessory subunits in native channels. Cells transfected with α2bδ alone did not bind IgG from any of the 3 patients (Results not shown.). However, they were clearly transfected as seen by GFP fluorescence as well as by binding of the specific anti-α2 subunit antibody (Results not shown.). In contrast, cells transfected with only the β3 subunit were detected by IgG from all 3 LEMS and SCLC but not the MG patient (Fig. 3). Figure 4 depicts results obtained from cells transfected with only a single type of α1
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LEMS IgG and Cav channel subunits, Page 12 subunit (α1A, α1B-1, α1C-1, or α1E). Although data shown are from a single patient, identical results were seen with all 3 LEMS patients’ IgG. There appeared to be differences in signal intensity and duration, but no attempt was made to quantitate the fluorescence. In any case, all 4 sets of transfectants showed positive fluorescent signal. Similar results occurred when transfected cells were probed with SCLC patient IgG (Results not shown.). However, the MG patient IgG produced no detectable signal in cells transfected with any of the subunit(s) (Results not shown.). Thus we conclude that autoantibodies from patients with LEMS and SCLC, but not MG, are directed against multiple α1 and β3 but not the α2δ subunit of voltage-sensitive VGCCs.
Discussion The initial humoral autoimmune response in LEMS patients is assumed to be generated against the VGCC subunit antigens on the lung carcinoma cells which over-express multiple subtypes of HVA VGCCs. Our results provide specific, direct immunocytological evidence for binding of voltage-sensitive VGCC subunits in the etiology of LEMS. Specifically, we have shown that: 1) IgG isolated from LEMS patient sera recognize more than 1 subunit of HVA VGCCs; 2) the antibodies from patient sera bind to HEK293 cells expressing VGCCs containing α1A, α1B, α1C, or α1E plus α2δ and β subunits as well as those expressing a single α1A, α1B , α1C, or α1E subunit; 3) despite its normal intracellular location, the β3 subunit is also recognized by the LEMS antibodies; 4) the α2δ subunit was not recognized by antibodies of the LEMS patients; 5) antibodies from a patient with MG did not recognize the proteins, whereas those from a patient with SCLC did. The ability of IgG from LEMS patients to bind with cells expressing functional VGCCs of all 4 subtypes suggests that antibodies are directed against a conserved region of the protein.
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LEMS IgG and Cav channel subunits, Page 13 However, this effect could also result from use of a constant β or α2δ subunit in all the transfections if autoantibodies targeted either β3 or α2δ. When we repeated the experiments using cells transfected with only 1 subunit, it became clear that the LEMS antibodies recognize all 4 α1 subunits. The most rational explanation for this observation is that circulating autoantibodies are generated against all extracellular antigens overexpressed on the SCLC cells as a primary response. There may be subtle differences in the type of response and the outcomes resulting therefrom. Pinto et al.,26 reported surface binding of LEMS patient IgG to both α1A and α1B in HEK293 cells stably expressing these channels when assayed using immunoprecipitation of radiolabelled ω-conotoxin MVIIC and ω-agatoxin GVIA respectively. However, functional disruption of only P/Q-type but not N-type current was observed by these authors. This makes sense in part, because the α1A subunit, which confers the P/Q subunit phenotype, is the primary VGCC subtype responsible for ACh release at the mammalian motor nerve terminal. However, anti N-type autoantibodies are known to exist in LEMS plasma,29,8,9,36 and dysautonomia is commonly reported in LEMS patients.10 Release of ACh at autonomic ganglion and postganglion effector junctions has a strong N-type HVA dependence. Giovanini et al.,37 and our own results38 demonstrated that in a mouse expressing genetically-based P/Q-type functional deficiency, both N- and R- type VGCCs can become involved in ACh release. The former study was done in the presence of the K+ channel blocker 4aminopyridine. Our study was not. Furthermore, the work from our laboratory21,22,39 as well as others,40,23 indicated that P/Q-type function can be substituted by L-type VGCCs in LEMS patients and in LEMS Ig-treated mice. The avidity and activity of antibodies directed against the P/Q-types of VGCCs can increase with disease progression and the P/Q-type function continues to be substituted by other subtypes of VGCCs as a survival mechanism. As these events
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LEMS IgG and Cav channel subunits, Page 14 progress, antibodies can develop against these substituted phenotypes of VGCCs as well. Therefore, autoantibodies against all the VGCCs types eventually develop. The difference in response could be due either to the sequence of events described above or purely a qualitative and circumstantial observation. Similarly, antibodies in LEMS patient sera against other components of the functional VGCC complex such as synaptotagmin41 or presynaptic muscarinic receptors42 may represent a secondary response. We propose that the anti-β subunit response in LEMS patients arises as a secondary response resulting from cell damage and death and the release of intracellular β subunit antigenic epitopes. Response against the β subunits is probably not a primary response because of its intracellular placement. Since we used only 1 isoform of the β subunit, we do not know if other isoforms will yield similar results. Similarly, it is difficult to predict how the several types of β subunits that result from splice variants of the 4 basic β1-4 subunit gene products would behave. As shown in studies in which mice express the lethargic (lh) mutation, in which β4 is trucated at the α1 interaction domain, other β subunits substitute to retain P/Q-channel function.43-45 Perhaps because of the multiplicity of β subunits, several could substitute the function of β subunits against which the immune response is generated initially. Therefore, at any given time in the life of a patient, antibodies against 1 or more of the β subunits may eventually develop. The lack of anti-α2δ subunit-responsive antibodies in LEMS patient IgG is perhaps to be expected and logical. As demonstrated in HIV research, immune responses are often weak or absent against highly conserved and critical gene products and heavily glycosylated proteins.46,47 The α2 subunit is one of the most highly conserved, common, and invariant moieties of any VGCC subtype; only 4 genetic variants have been identified so far.48-50 It is also heavily glycosylated.49 Whereas the function of 1 type of α1 or β subunit can often be substituted with
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LEMS IgG and Cav channel subunits, Page 15 another type in cases of compensation or adaptation, the α2 subunit function may not be substitutable.51 Therefore, even in the presence of an excess of over-expressed antigens that include the α2 subunit, autoantibodies are not generated against the α2 subunit. Other proteins including the synaptic vesicle release cycle protein syntaxin or M1 muscarinic receptors have also been suggested as possible targets of LEMS autoantibodies.41 Our results cannot discount that autoimmune responses are mounted to additional proteins in some or all patients, or indeed are universally produced. However, it is clear that the principal functional subunits of HVA VGCCs are definite targets for binding of autoantibodies in patients with LEMS. On the surface, the lack of binding of MG patient is to be expected, as the primary targets in MG are the muscle-type nicotine receptors. However, there are several clinical reports of patients with MG who express a mixed phenotype, so some small percentage of MG patients may also exhibit antibody binding.52-55 In conclusion, we provide direct immunocytochemical evidence for binding of IgG of 3 patients with LEMS to multiple subtypes of pore-forming subunits of HVA VGCCs. The antibody spectrum undoubtedly varies among patients, presumably related to the nature of the immune response to the tumor. The clinical course of the LEMS is clearly going to vary depending on the titer and avidity of the relative autoantibodies.
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LEMS IgG and Cav channel subunits, Page 16 Figure Legends Figure 1: HEK293 cells transfected with α1 subunit cDNA for specific VGCC subtypes show positive reactivity to their specific antibodies. In all figures, the left panel shows GFP fluorescence as a transfection reporter. In the right panels, the same microscopic field is shown with fluorescence from the label on secondary antibodies. No cross-reactivity was ever seen with a different subunit antibody (Results not presented.).
Figure 2: HEK293 cells transfected with full complement of specific VGCC subunits (α1, β3 and α2δ) show reactivity to LEMS patient immunoglobulins. Results are from one of the three patients tested, all positive. Because the GFP reporter cDNA was used at approximately 1/5 the amount of VGCC cDNA, there are many more transfected cells with positive secondary antibody fluorescence than with GFP fluorescence.
Figure 3: Cells transfected with only the β3 subunit of HVA VGCCs demonstrated binding of all LEMS and SCLC patients but not MG patient.
Figure 4: LEMS patient IgG showed reactivity to cells transfected with only a single type of α1 subunit for HVA VGCCs (α1A, α1B-1, α1C-1, or α1E). Although data are presented from only 1 patient, identical results were seen with all 3 LEMS patients IgG tested.
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LEMS IgG and Cav channel subunits, Page 17 Acknowledgments Supported by a grant from the Muscular Dystrophy Association of America (MDA176219) and by NINDS grant R01NS051833.
The word processing assistance of Beth Anne Hill and help with figure preparation by Jessica Hauptman is appreciated.
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LEMS IgG and Cav channel subunits, Page 18 Abbreviations DPBS=Dulbecco’s phosphate buffered saline GFP=green fluorescent protein HEK293=human embryonic kidney cells HEPES=4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HVA=high voltage-activated calcium current IgG=immunoglobulin G LEMS=Lambert-Eaton Myasthenic Syndrome LVA=low voltage-activated calcium current MG=myasthenia gravis SCLC=small cell lung carcinoma, also called oat cell carcinoma VGCCs=voltage-gated Ca2+ channels
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LEMS IgG and Cav channel subunits, Page 19 References 1. Lambert EH, Eaton LM, Rooke ED. Defect of neuromuscular conduction associated with malignant neoplasm. Am J Physiol 1956; 187: 612—613. 2. Vincent A, Lang B, Newsom-Davis J. Autoimmunity to the voltage-gated calcium channel underlies the Lambert-Eaton myasthenic syndrome, a paraneoplastic disorder. Trends Neurosci 1989; 12: 496—502. 3. Sher E, Carbone E, Clementi F. Neuronal calcium channels as target for Lambert-Eaton myasthenic syndrome autoantibodies. Ann NY Acad Sci 1993; 681: 373—381. 4. Katz E, Ferro PA, Weiss G, Uchitel OD. Calcium channels involved in synaptic transmission at the mature and regenerating mouse neuromuscular junction. J Physiol (Lond) 1996; 497: 687—689. 5. Hirning LD, Fox AP, McCleskey EW, Oliver BM, Thayer SA, Miller RJ, et al. Dominant role of N-type Ca2+ channels in evoked release of norepinephrine from sympathetic neurons. Science 1998; 239: 57—61. 6. Lemos JR, Nowycky MC. Two types of calcium channels coexist in peptide-releasing vertebrate nerve terminals. Neuron 1989; 2: 1419—1426. 7. Owen PJ, Marriott DB, Boarder MR. Evidence for a dihydrophyridine-sensitive and conotoxin-insensitive release of noradrenaline and uptake of calcium in adrenal chromaffin cells. Br J Pharmacol 1989; 97: 133—138. 8. Waterman SA. Role of N-, P-, and Q-type voltage-gated calcium channels in transmitter release from sympathetic neurones in the mouse isolated vas deferens. Br J Pharmacol 1997; 120: 393—398. 9. Waterman SA. Voltage-gated calcium channels in autonomic neuroeffector transmission.
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LEMS IgG and Cav channel subunits, Page 20 Prog Neurobiol 2000; 60: 181—210. 10. Waterman SA. Autonomic dysfunction in Lambert-Eaton myasthenic syndrome. Clin Auton Res 2001; 11: 145—154. 11. Benatar M, Blaes F, Johnston I, Wilson K, Vincent A, Beeason D, et al. Presynaptic neuronal antigens expressed by a small cell lung carcinoma cell line. J Neuroimmunol 2001; 113: 153—162. 12. Sabater L, Titulaer M, Saiz A, Verschuuren J, Gure AO, Graus, F. SOX1 antibodies are markers of paraneoplastic Lambert-Eaton myasthenic syndrome. Neurology 2008; 70: 924–928. 13. Takamori M, Maruta T, Komai K. Lambert-Eaton myasthenic syndrome as an autoimmune calcium channelopathy. Neurosci Res 2000; 36: 183—191. 14. Cull-Candy SG, Miledi R, Trautmann A, Uchitel OD. On the release of transmitter at normal, myasthenia gravis and myasthenic syndrome affected human end-plates. J Physiol (Lond) 1980; 299: 621—638. 15. Lambert EH, Elmqvist D. Quantal components of end-plate potentials in the myasthenic syndrome. Ann NY Acad Sci 1971; 183: 183—199. 16. Lang B, Newsom-Davis J, Prior C, Wray D. Antibodies to motor nerve terminals: an electrophysiological study of a human myasthenic syndrome transferred to mouse. J Physiol (Lond) 1983; 344: 335—345. 17. Kim YI. Lambert-Eaton Myasthenic Syndrome: evidence for calcium channel blockade. Ann NY Acad Sci 1987; 505: 377—379. 18. Kim YI, Neher E. IgG from patients with Lambert-Eaton syndrome blocks voltagedependent calcium channels. Science 1998; 239: 405—408.
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LEMS IgG and Cav channel subunits, Page 21 19. Roberts A, Pereira S, Lang B, Vincent A, Newsom-Davis J. Paraneoplastic myasthenic syndrome IgG inhibits
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Ca2+ flux in a human small cell carcinoma line. Nature 1985;
317: 737—739. 20. Peers C, Lang B, Newsom-Davis J, Wray DW. Selective action of myasthenic syndrome antibodies on calcium channels in a rodent neuroblastoma x glioma cell line. J Physiol (Lond) 1990; 421: 293—308. 21. Flink MT, Atchison WD. Passive transfer of Lambert-Eaton syndrome to mice induces dihydropyridine sensitivity of neuromuscular transmission. J Physiol (Lond) 2002; 543: 567—576. 22. Smith DO, Conklin MW, Jensen PJ, Atchison WD. Decreased calcium currents in motor nerve terminals of mice with Lambert-Eaton myasthenic syndrome. J Physiol (Lond) 1995; 487: 115—123. 23. Garcia KD, Beam KG. Reduction of calcium currents by Lambert-Eaton syndrome sera: motoneurons are preferentially affected, and L-type currents are spared. J Neurosci 1996; 16: 4903—4913. 24. Garcia KD, Mynlieff M, Sanders DB, Beam KG, Walrond JP. Lambert-Eaton sera reduce low-voltage and high-voltage activated Ca2+ currents in murine dorsal root ganglion neurons. Proc Natl Acad Sci USA 1996; 93: 9264—9269. 25. Pinto A, Gillard S, Moss F, Whyte K, Brust P, Williams M, et al. Human autoantibodies specific for the α1A calcium channel subunit reduce both P-type and Q-type calcium currents in cerebellar neurons. Proc Natl Acad Sci USA 1998; 95: 8328—8333. 26. Pinto A, Iwasa K, Newland C, Newsom-Davis J, Lang B. The action of Lambert-Eaton myasthenic syndrome immunoglobulin G on cloned human voltage-gated calcium
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LEMS IgG and Cav channel subunits, Page 22 channels. Muscle Nerve 2002; 25: 715—724. 27. Rosenfeld MR, Wong E, Dalmau J, Manley G, Posner JB, Sher E, et al. Cloning and characterization of a Lambert-Eaton myasthenic syndrome antigen. Ann Neurol 1993; 33: 113—120. 28. Veschuuren JJ, Dalmau J, Tunkel R, Lang B, Graus F, Schramm L, et al. Antibodies against the calcium channel beta-subunit in Lambert-Eaton myasthentic syndrome. Neurology 1998; 50: 475—479. 29. Iwasa K, Pinto A, Vincent A, Lang B. LEMS IgG binds to extracellular determinants on Ntype voltage-gated calcium channels, but does not reduce VGCC expression. Ann NY Acad Sci 2003; 998: 196—199. 30. Lang B, Pinto A, Giovannini F, Newsom-Davis J, Vincent A. Pathogenic autoantibodies in the Lambert-Eaton myasthenic syndrome. Ann NY Acad Sci 2003; 998: 187—195. 31. Hans M, Urrutia A, Deal C, Brust PF, Stauderman K, Ellis SB, et al. Structural elements in domain IV that influence biophysical and pharmacological properties of human α1Acontaining high-voltage-activated calcium channels. Biophys J 1999; 76: 1384—1400. 32. Williams ME, Brust PF, Feldman DH, Patthi S, Simerson S, Maroufi A, et al. Structure and functional expression of an omega-conotoxin-sensitive human N-type calcium channel. Science 1992a; 257: 389—395. 33. Williams ME, Marubio LM, Deal CR, Hans M, Brust PF, Philipson LH, et al. Structure and functional characterization of neuronal α1E calcium channel subtypes. J Biol Chem 1994; 269: 22347—22357. 34. Williams ME, Feldman DH, McCue AF, Brenner R, Velicelebi G, Ellis SB, et al. Structure and functional expression of α1, α2, and β subunits of a novel human neuronal calcium
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LEMS IgG and Cav channel subunits, Page 23 channel subtype. Neuron 1992b; 8: 71—84. 35. Peng S, Hajela RK, Atchison WD. Characteristics of block by Pb2+ of function of human neuronal L-, N-, and R-type Ca2+ channels transiently expressed in human embryonic kidney 293 cells. Mol Pharmacol 2002; 62: 1418—1430. 36. Wirtz PW, Roep BO, Schreuder GM, van Doorn PA, van Engelen BG, Kuks JB, et al. HLA class I and II in Lambert-Eaton myasthenic syndrome without associated tumor. Human Immunol 2001b; 62: 809—813. 37. Giovannini F, Sher E, Webster R, Boot J, Lang B. Calcium channel subtypes contributing to acetylcholine release from normal, 4-amino-pyridine-treated and myasthenic syndrome auto-antibodies-affected neuromuscular junctions. Br J Pharmacol 2002; 136: 1135— 1145. 38. Pardo NE, Hajela RK, Atchison WD. Acetylcholine release at neuromuscular junctions of adult tottering mice is controlled by N-(Cav2.2) and R-type (Cav2.3) but not L-type (Cav1.2) Ca2+ channels. J Pharmacol Exp Ther 2006; 319: 1009—1020. 39. Xu YF, Hewlett SJ, Atchison WD. Passive transfer of Lambert-Eaton myasthenic syndrome induces dihydrophyridine sensitivity of ICa in mouse motor nerve terminals. J Neurophysiol 1998; 80: 1056—1069. 40. Campbell DB, Hess EJ. L-type calcium channels contribute to the tottering mouse dystonic episodes. Mol Pharmacol 1999; 55: 23—31. 41. Leveque C, Hoshino T, David P, Shoji-Kasai Y, Leys K, Omori A, et al. The synaptic vesicle protein synaptotagmin associates with calcium channels and is a putative Lambert-Eaton myasthenic syndrome antigen. Proc Natl Acad Sci USA 1992; 89: 3625—3629. 42. Takamori M, Motomura M, Fukudome T, Yoshikawa H. Autoantibodies against M1
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LEMS IgG and Cav channel subunits, Page 24 muscarinic acetylcholine receptor in myasthenic disorders. Eur J Neurol 2007; 14: 1230—1235. 43. Vance CL, Begg CM, Lee WL, Haase H, Copeland TD, McEnery MW. Differential expression and association of calcium channel α1B and β subunits during rat brain ontogeny. J Biol Chem 1998; 273: 14495—14502. 44. McEnery MW, Copeland TD, Vance CL. Altered expression and assembly of N-type calcium channel α1B and β subunits in epileptic lethargic (lh) mouse. J Biol Chem 1998; 273: 21435—21438. 45. Burgess DL, Biddlecome GH, McDonough SI, Diaz ME, Zilinski CA, Bean BP, Campbell KP, Noebels JL. β subunit reshuffling modifies N- and P/Q- type Ca2+ channel subunit compositions in lethargic mouse brain. Mol Cell Neurosci 1999; 13: 293—311. 46. Burton DR. A vaccine for HIV type 1: the antibody perspective. Proc Natl Acad Sci USA 1997; 94: 10018—10023. 47. VanCott TC, Betake FR, Burke DS, Redfield RR, Birx DL. Lack of induction of antibodies specific for conserved, discontinuous epitopes of HIV-1 envelope glycoprotein by candidate AIDS vaccines. J Immunol 1995; 155: 4100—4110. 48. Gao B, Sekido Y, Maximov A, Saad M, Forgacs E, Latif F, et al. Functional properties of a new voltage-dependent calcium channel α2δ auxiliary subunit gene (CACANA2D2). J Biol Chem 200; 275: 12237—12242. 49. Klugbauer N, Lacinova L, Marais E, Hobom M, Hofmann F. Molecular diversity of the calcium channel α2δ subunit. J Neurosci 1999; 19: 684—691. 50. Qin N, Yagel S, Momplaisir ML, Codd EE, D’Andrea MR. Molecular cloning and characterization of the human voltage-gated calcium channel α2δ4 subunit. Mol
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LEMS IgG and Cav channel subunits, Page 25 Pharmacol 2002; 62: 485—496. 51. Arikanth J, Campbell KP. Auxiliary subunits: essential components of the voltage gated calcium channel complex. Curr Opin Neurobiol 2003; 13: 298—307. 52. Oh SJ, Sher E. MG and LEMS overlap syndrome: case report with electrophysiological and immunological evidence. Clin Neurophysiol 2005; 116: 1167—1171. 53. Roohi F, Smith PR, Bergman M, Baig MA, Sclar G. A diagnostic and management dilemma: combined paraneoplastic myasthenia gravis and Lambert-Eaton myasthenic syndrome presenting as acute respiratory failure. Neurologist 2006; 12: 322—326. 54. Kim JA, Lim YM, Jang EH, Kim KK. A patient with coexisting myasthenia gravis and lambert-eaton myasthenic syndrome. J Clin Neurol 2012; 8: 235—237. 55. Sha SJ, Layzer RB. Myasthenia gravis and Lambert-Eaton myasthenic syndrome in the same patient. Muscle Nerve 2007; 36: 115—117.
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HEK293 cells transfected with specific α1 subunit show positive reactivity to their specific antibodies. In all figures, the left panel shows GFP fluorescence as a transfection reporter. In the right panels, the same microscopic field is shown with fluorescence from the label on secondary antibodies. No cross-reactivity was ever seen with a different subunit antibody (results not presented). 176x231mm (300 x 300 DPI)
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HEK293 cells transfected with full complement of Ca2+ channel subunits (α1, β3 and α2δ) show reactivity to LEMS patient immunoglobulins. Results are from one of the three patients tested, all positive. Because the GFP reporter cDNA was used at approximately 1/5 the amount of Ca2+ channel cDNA, there are many more transfected cells with positive secondary antibody fluorescence than with GFP fluorescence. 165x181mm (300 x 300 DPI)
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Cells transfected with only the β3 subunit demonstrated binding of all LEMS and SCLC patients but not MG patient. 167x253mm (300 x 300 DPI)
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LEMS patient IgG showed reactivity to cells transfected with only a single type of α1 subunit (α1A, α1B-1, α1C-1, or α1E). Although data are presented from only one patient, identical results were seen with all three LEMS patients IgG tested. 153x182mm (300 x 300 DPI)
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Ravindra K. Hajela, Ph.D., Kristin M. Huntoon, D.O, Ph.D., and William D. Atchison, Ph.D.
Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan, USA.
Address all correspondence including reprint requests to: Dr. William D. Atchison Department of Pharmacology and Toxicology Michigan State University 1355 Bogue Street B331 Life Sciences Building East Lansing, MI 48824-1317 phone: (517) 353-4947 fax: (517) 432-1341 e-mail: [email protected]
Running Title: LEMS IgG and Cav channel subunits Dr. Huntoon’s Current Address: Surgical Neurology, National Institute of Neurological Disorders and Stroke, National Institute of Health, Bethesda, Maryland
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/mus.24295
Muscle & Nerve
LEMS IgG and Cav channel subunits, Page 2 Abstract Introduction: Lambert Eaton Myasthenic syndrome (LEMS) is an autoimmune presynaptic neuromuscular disorder. Autoantibodies against subunits of voltage gated calcium channels (VGCCs) associated with acetylcholine release are thought to cause LEMS. Methods: HEK293 cells expressing specific individual recombinant subunits of α1A, B, C and E, β3, and α2δ of human neuronal VGCCs were exposed to antibodies from 3 LEMS patients, a patient with small cell lung carcinoma, and 1 with myasthenia gravis. Results: All LEMS patient antibodies bound to cells containing any of the α1 or β3 alone or combined with α2δ subunits, but not α2δ alone. Autoantibodies from the patient with small cell lung carcinoma but not the myasthenia gravis patient targeted the same VGCC subunits. Conclusion: autoantibodies from LEMS patients bind directly to multiple VGCC α1 subunits as well as the β3 subunit. Thus, multiple components of the presynaptic VGCC complex are prospective targets for antibodies in LEMS.
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LEMS IgG and Cav channel subunits, Page 3 Keywords: 1. Lambert-Eaton Myasthenic Syndrome 2. Voltage-gated Ca2+ Channels 3. Neuromuscular Disease 4. Small Cell Lung Carcinoma 5. Acetylcholine Release
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LEMS IgG and Cav channel subunits, Page 4 Introduction Lambert-Eaton Myasthenic Syndrome (LEMS) is a paraneoplastic, autoimmune disorder of neuromuscular transmission. It results in skeletal muscle weakness which involves the lower more than upper extremities1. Muscle weakness is due to a reduction in the number of quanta of acetylcholine (ACh) released by the motor nerve terminal in response to a nerve impulse.2 Clinical manifestations of the disease are ascribed to disruption of Ca2+ influx through voltagegated Ca2+ channels (VGCCs) in the motor nerve terminal, a process essential for release of ACh.3 Although there are various subtypes of VGCCs, the P/Q-type (Cav2.1) are involved primarily in ACh release from adult mammalian motor nerve terminals.4 Other Ca2+ channel phenotypes, such as N-type (Cav2.2) and L-type (Cav1.3), are responsible for transmitter release from peripheral autonomic nerve and central terminals or hormone release respectively,5-10 and some are also affected in LEMS10. Approximately two-thirds of LEMS patients also have small cell lung carcinoma (SCLC) in which the cancer cells overexpress several types of VGCCs.11 LEMS is thought to be triggered by cross-reactive binding of circulating autoantibodies to motor nerve terminals, presumably produced initially in response to the VGCC complex components. Conceptually, autoantibodies against any of the several proteins that form a functional VGCC complex, or proteins that present these antigens to the immune system, can be generated and found in patient sera. Indeed evidence for all of these possibilities has been presented. For example, approximately 64% of paraneoplastic LEMS patients with SCLC also have antibodies against SOX1, a transcription factor protein strongly expressed in the developing nervous system, suggesting the possibility of a very early marker of the future predisposition to LEMS/SCLC.12 All the present evidence suggests that antibodies against proteins closely linked to the VGCC
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LEMS IgG and Cav channel subunits, Page 5 functional complex could be found in some patients and not others.13 Therefore, the circulating autoantibodies against components of VGCCs are prime candidates for disrupting motor nerve terminal function.14-18,2 However, whether antibodies are generated against more than 1 specific subtype of VGCC or multiple subunits of those channels is not yet known with certainty. In vitro studies of Ca2+ influx during depolarization19 or measurement of inward currents through VGCCs using divalent charge carriers show reduction when cells in culture (including those of neuronal origin or small cell lung carcinoma) are exposed to LEMS antibodies.18-20 Perineurial-generated Ca2+ currents from nerve terminals of mice treated chronically with LEMS patient sera or plasma are reduced in amplitude in general but show an apparent upregulation of L-type current and dihydropyridine sensitivity of neuromuscular transmission.21,22 Both low voltage-activated (LVA) and high voltage-activated (HVA) Ca2+ currents were reduced in amplitude in dorsal root ganglia from mice treated with LEMS patient sera, but currents were reduced preferentially in motor neurons compared to sensory neurons in culture, and L-type currents were spared.23,24 However, in HEK293 cells stably expressing Cav2.1 (P/Q-type), Cav2.2 (N-type), Cav1.2 (L-type), and Cav2.3 (R-type) VGCCs, LEMS patient IgG caused a significant reduction in K+-stimulated increase of Ca2+i only in Cav2.1 (P/Q-type)-expressing cells.25,26 The intracellular β subunits have also been implicated in the etiology of LEMS, because a screen of an expression cDNA library using LEMS sera resulted in isolation of a β subunit cDNA clone.27 Recognition of the protein in western blots produced expression of the cDNA clone of the β subunit.28 Results of prior pharmacological studies, toxin binding, current recording, or other indirect studies29,30,25,26, taken together, demonstrate that 1 or more VGCC subunits can be targeted by LEMS antibodies. However, no cytological evidence using antibodies from LEMS
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LEMS IgG and Cav channel subunits, Page 6 patient sera or plasma has been presented. In this study, we provide direct immunocytological evidence that multiple VGCC subunits can bind circulating autoantibodies from LEMS patients and an SCLC patient, but not a myasthenia gravis (MG) patient. In order to determine which individual subtypes or subunits of VGCCs are recognized by the circulating autoantibodies in the LEMS patient sera and avoid confounding effects associated with the presence of the secretory complex or other potential targets, we used a heterologous system transiently expressing cDNA clones of specific subunits of human neuronal HVA VGCCs in HEK293 cells in culture.
Materials, Methods and Experimental Design HEK293 cells #CRL-1573 were purchased from American Type Culture Collection (Rockville, MD). Expression cDNA clones for human neuronal VGCC α1A-2 (from cerebellum31), α1B-1 (from IMR32 cells of human neuronal origin),32 α1C-1 (from hippocampus, personal communication, Dr. Mark Williams, Merck Research Laboratories) or α1E-3 (from hippocampus33), and α2bδ (from brain stem and basal ganglia34) and β3-a (from hippocampus, personal communications, Dr. Mark Williams, Merck Research Laboratories) subunits were provided by Dr. Kenneth Stauderman of SIBIA Neurosciences, now Merck Research Laboratories, San Diego, CA. The LEMS, SCLC, and MG patient sera for IgG preparation were provided by Drs Andrew Massey, University of Kentucky Medical Center, Lexington, KY, Eva Feldman and James Albers, University of Michigan Medical Center, Ann Arbor, MI, and Shin Oh, University of Alabama Medical Center, Birmingham, AL. Control human plasma was obtained as outdated donated material from the blood bank of the American Red Cross, Lansing, MI, courtesy of Dr. Donald Penner. All sera/plasma were supplied to us with patient age and gender as the only identifiers. Samples of LEMS, SCLC, and MG plasma were collected during
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LEMS IgG and Cav channel subunits, Page 7 the course of routine plasma exchange therapy with normal informed consent of the patients in accordance with the approval of regulatory Human Subject Committees of the respective institutions. All reagents were off the shelf pure or ultra pure laboratory grade unless specifically noted. HEK293 cells were grown at 37oC in Eagle Modified Essential Medium fortified with 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 10% (w/v) fetal bovine serum, and penicillin, streptomycin, antimycotic, and antibiotics (GIBCO-BRL/Invitrogen Technologies, Gaithersburg, MD) in a 5% CO2 environment. One day before gene transfer, cells were plated at a density of 5x105 on poly Llysine-coated 25 mm round cover slips in 35 mm culture dishes. Cells were transfected with a mixture of plasmids containing expression cDNA clones of human neuronal VGCC subunits α1A2,
α1B-1, α1C-1, or α1E-3, either individually or together with α2bδ and β3-a and a green fluorescent
protein (GFP) cDNA clone, using Fugene 6 (Roche Molecular Biochemicals, Indianapolis, IN) and following the manufacturer’s instructions. Reactions contained a total of 3 µl of Fugene 6 and 1 µg plasmid DNA containing the 3 VGCC subunits in 1:1:1 molar ratio, or only the specific subunit. GFP plasmid was also included at 20% of the total DNA. Two to 3 days were allowed for optimal transient expression of proteins, at which time the cells were examined for GFP expression using an Olympus IX70 fluorescence microscope (Olympus Optical, Tokyo, Japan) with a mercury arc burner at excitation wavelength of 460-490 nm and emission wavelength of 510-550 nm for GFP expression and excitation wavelength of 510-550 nm and emission wavelength of 580-590 nm for rhodamine fluorescence. LEMS patient IgG samples were purified using affinity column chromatography and antihuman IgG sepharose or agarose beads (Sigma-Aldrich, St. Louis, MO). Protein content was
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LEMS IgG and Cav channel subunits, Page 8 quantitated using the Bio-Rad DC protein assay kit (Bio Rad, Hercules, CA) with bovine serum albumin as a standard. For immunocytochemistry, each cover slip was rinsed 3 times (3 min/rinse) with Dulbecco phosphate buffered saline (DPBS) in (mM): CaCl2 45, KCl 1.34, KH2PO4 0.74, MgCl2 0.25, NaCl 70, NaH2PO4 4, (pH at 7.4). Cells were fixed with 4% (w/v) paraformaldehyde in DPBS (2 ml/cover slip) for 10 min at room temperature of 23-25oC followed by another set of rinses as above. Fixed cells were permeabilized by soaking the cover slips in 0.2% (w/v) Triton X-100 in DPBS for 10 min at room temperature. Following a set of 3 rinses as above, the cover slips were incubated for 10 min at room temperature in a blocking solution. In experiments using patient IgG, the blocking solution contained 7.8% (w/v) goat serum, 5.5% (w/v) horse serum, and 35.25 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] in DPBS. For experiments using anti-subunit antibodies, blocking solution contained 3% (w/v) bovine serum albumin (Sigma-Aldrich, St. Louis, MO) in DPBS. After blocking, the cover slips were incubated in 1.2 ng/ml of LEMS/MG/SCC IgG or anti-subunit antibodies (Alomone Labs Ltd., Jerusalem, Israel) made up in blocking solution and turned inverted on cavity slides (Fisher Scientific, Itasca, IL). These slides were incubated in a dark, humidified chamber for 1 hr at room temperature, then for 30 hrs at 4oC. Cells were subsequently rinsed 3 times as before, followed by incubation in 1:5000 dilution of rhodamine red-X-conjugated donkey anti-human IgG (Jackson Immuno Research Laboratories Inc., West Grove, PA) or rhodamine-conjugated donkey anti-rabbit IgG antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), respectively. Cover slips were gently rinsed again 3 times with blocking solution before examination under the fluorescence microscope. No attempt was made to quantitate the epifluorescence. Two sets of experiments were performed. In the first, fully functional VGCCs of
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LEMS IgG and Cav channel subunits, Page 9 predetermined subtype were expressed in HEK293 cells using an equimolar combination of a specific α1 subunit with constant α2δ and β subunits. Functional expression of the specific subtype has previously been demonstrated using whole cell recordings.35 The second set of experiments was designed to determine which subunit(s) were specifically recognized by the LEMS antibodies. cDNA of only 1 subunit was used in any given transfection. In all experiments, the cells were fixed and permeabilized before performing the immunocytological screening. Permeabilization was done to facilitate antibody accessibility to target VGCC subunits in the cytoplasm. Each experiment included various sets of positive and negative controls. To conserve space, most of these results are not shown in the manuscript. One set of cover slips with specific subunit-transfected cells was treated with commercially available anti-subunit-specific primary antibodies generated in rabbits in order to verify the protein expression and to establish binding of antibody. These cover slips were treated further with rhodamine-conjugated donkey antirabbit secondary antibody to visualize the binding of the primary antibody. Cover slips treated with either only anti-subunit specific primary antibody, with only rhodamine-conjugated donkey anti-rabbit secondary antibody, or only rabbit serum and the labeled secondary antibody never showed any fluorescence (Results not shown). This assured us that any signal visualized and recorded was due to binding of the labeled secondary antibodies to the anti-subunit specific antibody bound to the VGCCs expressed in the transfected cells growing on the cover slips. Specificity of anti-subunit antibodies was tested by treating cells with each of the anti-subunit antibodies separately as well as treating the cells with antibodies for subunits other than those with which the cells were transfected. No cross-reactivity was ever seen among subunit subtypespecific antibodies. (Results not shown.) Cells grown on poly L-lysine-treated test cover slips
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LEMS IgG and Cav channel subunits, Page 10 were treated with LEMS patient IgG followed by rhodamine-conjugated donkey anti-human secondary antibodies. Negative controls for this set of experiments included incubation with only LEMS patient IgG, only the labeled secondary antibodies, and control human serum plus secondary antibodies. No fluorescence was seen in any of the controls (Results not shown.). Finally, untransfected cells were treated with patient IgG or anti-subunit antibody and respective labeled secondary antibody; they exhibited no binding or autofluorescence.
Results Figures 1-4 depict epifluorescence generated in HEK cells either transfected with single specific subunits of HVA VGCCs, whole channel complexes, or controls. In each figure, the left panel depicts GFP fluorescence, and the same optical field in the right panel depicts fluorescence due to binding of specific secondary antibody. Often, greater numbers of cells are seen as antibody positive than as GFP positive. This is due to the fact that only 20% of transfecting DNA used was for GFP, giving the statistical probability of 4 non-green cells being transfected with VGCC subunit DNA for every cell transfected with GFP. The different pattern of fluorescence in different cells is due to the distribution of the target proteins variously in the cytoplasm and the membrane. The punctate signal on the perimeter represents the membrane-bound signal, while the general diffuse signal is from the top, the bottom, and the cytoplasm of the cell. The apparent opacity is the very dense nuclear area. Subunits not yet fully processed would be found in the cytoplasm. Cytoplasmic localization would also occur when only a single subunit was transfected, because the transport and proper insertion of the channel in the cell membrane is highly dependent on accessory subunits, especially the β subunit. Figure 1 demonstrates the binding of subunit-specific antibodies to cells transfected with
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LEMS IgG and Cav channel subunits, Page 11 specific subunits to serve as a positive control of expression of transfected cDNAs. Also included in the control experiments were assays to check for nonspecific binding of either the wrong antibody or only the secondary antibody to transfected cells and LEMS patient IgG and secondary antibody to nontransfected cells. Figure 2 shows the binding of IgG from 1 representative LEMS patient to cells transfected with the full complement of cDNA for all 3 subunits of VGCCs including 1 of the subunits α1A-2, α1B-1, α1C-1, or α1E-3 together with α2bδ and β3-a. Affinity-purified IgG from all 3 LEMS patients showed binding to cells transfected with each of the 4 sub-types of VGCCs. (Results only shown for a representative patient.) These results provide direct evidence that LEMS patient autoantibodies are directed against multiple subtypes or multiple subunits of VGCCs. It has been shown previously that LEMS patient IgGs immunoprecipitate more than 1 type of VGCC from several kinds of human and rat cells. However, Pinto et al., 26 and Sher et al.,3 reported that functions of only P/Q-type VGCCs are impaired in HEK293 cells stably transfected with expression cDNA clones. These contrasting observations, although obtained using different techniques, may be due to differences in sensitivity of techniques or use of a common α2δ and β3 subunit with different α1 subunits in heterologous expression experiments such as ours and a different complement of these accessory subunits in native channels. Cells transfected with α2bδ alone did not bind IgG from any of the 3 patients (Results not shown.). However, they were clearly transfected as seen by GFP fluorescence as well as by binding of the specific anti-α2 subunit antibody (Results not shown.). In contrast, cells transfected with only the β3 subunit were detected by IgG from all 3 LEMS and SCLC but not the MG patient (Fig. 3). Figure 4 depicts results obtained from cells transfected with only a single type of α1
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LEMS IgG and Cav channel subunits, Page 12 subunit (α1A, α1B-1, α1C-1, or α1E). Although data shown are from a single patient, identical results were seen with all 3 LEMS patients’ IgG. There appeared to be differences in signal intensity and duration, but no attempt was made to quantitate the fluorescence. In any case, all 4 sets of transfectants showed positive fluorescent signal. Similar results occurred when transfected cells were probed with SCLC patient IgG (Results not shown.). However, the MG patient IgG produced no detectable signal in cells transfected with any of the subunit(s) (Results not shown.). Thus we conclude that autoantibodies from patients with LEMS and SCLC, but not MG, are directed against multiple α1 and β3 but not the α2δ subunit of voltage-sensitive VGCCs.
Discussion The initial humoral autoimmune response in LEMS patients is assumed to be generated against the VGCC subunit antigens on the lung carcinoma cells which over-express multiple subtypes of HVA VGCCs. Our results provide specific, direct immunocytological evidence for binding of voltage-sensitive VGCC subunits in the etiology of LEMS. Specifically, we have shown that: 1) IgG isolated from LEMS patient sera recognize more than 1 subunit of HVA VGCCs; 2) the antibodies from patient sera bind to HEK293 cells expressing VGCCs containing α1A, α1B, α1C, or α1E plus α2δ and β subunits as well as those expressing a single α1A, α1B , α1C, or α1E subunit; 3) despite its normal intracellular location, the β3 subunit is also recognized by the LEMS antibodies; 4) the α2δ subunit was not recognized by antibodies of the LEMS patients; 5) antibodies from a patient with MG did not recognize the proteins, whereas those from a patient with SCLC did. The ability of IgG from LEMS patients to bind with cells expressing functional VGCCs of all 4 subtypes suggests that antibodies are directed against a conserved region of the protein.
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LEMS IgG and Cav channel subunits, Page 13 However, this effect could also result from use of a constant β or α2δ subunit in all the transfections if autoantibodies targeted either β3 or α2δ. When we repeated the experiments using cells transfected with only 1 subunit, it became clear that the LEMS antibodies recognize all 4 α1 subunits. The most rational explanation for this observation is that circulating autoantibodies are generated against all extracellular antigens overexpressed on the SCLC cells as a primary response. There may be subtle differences in the type of response and the outcomes resulting therefrom. Pinto et al.,26 reported surface binding of LEMS patient IgG to both α1A and α1B in HEK293 cells stably expressing these channels when assayed using immunoprecipitation of radiolabelled ω-conotoxin MVIIC and ω-agatoxin GVIA respectively. However, functional disruption of only P/Q-type but not N-type current was observed by these authors. This makes sense in part, because the α1A subunit, which confers the P/Q subunit phenotype, is the primary VGCC subtype responsible for ACh release at the mammalian motor nerve terminal. However, anti N-type autoantibodies are known to exist in LEMS plasma,29,8,9,36 and dysautonomia is commonly reported in LEMS patients.10 Release of ACh at autonomic ganglion and postganglion effector junctions has a strong N-type HVA dependence. Giovanini et al.,37 and our own results38 demonstrated that in a mouse expressing genetically-based P/Q-type functional deficiency, both N- and R- type VGCCs can become involved in ACh release. The former study was done in the presence of the K+ channel blocker 4aminopyridine. Our study was not. Furthermore, the work from our laboratory21,22,39 as well as others,40,23 indicated that P/Q-type function can be substituted by L-type VGCCs in LEMS patients and in LEMS Ig-treated mice. The avidity and activity of antibodies directed against the P/Q-types of VGCCs can increase with disease progression and the P/Q-type function continues to be substituted by other subtypes of VGCCs as a survival mechanism. As these events
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LEMS IgG and Cav channel subunits, Page 14 progress, antibodies can develop against these substituted phenotypes of VGCCs as well. Therefore, autoantibodies against all the VGCCs types eventually develop. The difference in response could be due either to the sequence of events described above or purely a qualitative and circumstantial observation. Similarly, antibodies in LEMS patient sera against other components of the functional VGCC complex such as synaptotagmin41 or presynaptic muscarinic receptors42 may represent a secondary response. We propose that the anti-β subunit response in LEMS patients arises as a secondary response resulting from cell damage and death and the release of intracellular β subunit antigenic epitopes. Response against the β subunits is probably not a primary response because of its intracellular placement. Since we used only 1 isoform of the β subunit, we do not know if other isoforms will yield similar results. Similarly, it is difficult to predict how the several types of β subunits that result from splice variants of the 4 basic β1-4 subunit gene products would behave. As shown in studies in which mice express the lethargic (lh) mutation, in which β4 is trucated at the α1 interaction domain, other β subunits substitute to retain P/Q-channel function.43-45 Perhaps because of the multiplicity of β subunits, several could substitute the function of β subunits against which the immune response is generated initially. Therefore, at any given time in the life of a patient, antibodies against 1 or more of the β subunits may eventually develop. The lack of anti-α2δ subunit-responsive antibodies in LEMS patient IgG is perhaps to be expected and logical. As demonstrated in HIV research, immune responses are often weak or absent against highly conserved and critical gene products and heavily glycosylated proteins.46,47 The α2 subunit is one of the most highly conserved, common, and invariant moieties of any VGCC subtype; only 4 genetic variants have been identified so far.48-50 It is also heavily glycosylated.49 Whereas the function of 1 type of α1 or β subunit can often be substituted with
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LEMS IgG and Cav channel subunits, Page 15 another type in cases of compensation or adaptation, the α2 subunit function may not be substitutable.51 Therefore, even in the presence of an excess of over-expressed antigens that include the α2 subunit, autoantibodies are not generated against the α2 subunit. Other proteins including the synaptic vesicle release cycle protein syntaxin or M1 muscarinic receptors have also been suggested as possible targets of LEMS autoantibodies.41 Our results cannot discount that autoimmune responses are mounted to additional proteins in some or all patients, or indeed are universally produced. However, it is clear that the principal functional subunits of HVA VGCCs are definite targets for binding of autoantibodies in patients with LEMS. On the surface, the lack of binding of MG patient is to be expected, as the primary targets in MG are the muscle-type nicotine receptors. However, there are several clinical reports of patients with MG who express a mixed phenotype, so some small percentage of MG patients may also exhibit antibody binding.52-55 In conclusion, we provide direct immunocytochemical evidence for binding of IgG of 3 patients with LEMS to multiple subtypes of pore-forming subunits of HVA VGCCs. The antibody spectrum undoubtedly varies among patients, presumably related to the nature of the immune response to the tumor. The clinical course of the LEMS is clearly going to vary depending on the titer and avidity of the relative autoantibodies.
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LEMS IgG and Cav channel subunits, Page 16 Figure Legends Figure 1: HEK293 cells transfected with α1 subunit cDNA for specific VGCC subtypes show positive reactivity to their specific antibodies. In all figures, the left panel shows GFP fluorescence as a transfection reporter. In the right panels, the same microscopic field is shown with fluorescence from the label on secondary antibodies. No cross-reactivity was ever seen with a different subunit antibody (Results not presented.).
Figure 2: HEK293 cells transfected with full complement of specific VGCC subunits (α1, β3 and α2δ) show reactivity to LEMS patient immunoglobulins. Results are from one of the three patients tested, all positive. Because the GFP reporter cDNA was used at approximately 1/5 the amount of VGCC cDNA, there are many more transfected cells with positive secondary antibody fluorescence than with GFP fluorescence.
Figure 3: Cells transfected with only the β3 subunit of HVA VGCCs demonstrated binding of all LEMS and SCLC patients but not MG patient.
Figure 4: LEMS patient IgG showed reactivity to cells transfected with only a single type of α1 subunit for HVA VGCCs (α1A, α1B-1, α1C-1, or α1E). Although data are presented from only 1 patient, identical results were seen with all 3 LEMS patients IgG tested.
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LEMS IgG and Cav channel subunits, Page 17 Acknowledgments Supported by a grant from the Muscular Dystrophy Association of America (MDA176219) and by NINDS grant R01NS051833.
The word processing assistance of Beth Anne Hill and help with figure preparation by Jessica Hauptman is appreciated.
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LEMS IgG and Cav channel subunits, Page 18 Abbreviations DPBS=Dulbecco’s phosphate buffered saline GFP=green fluorescent protein HEK293=human embryonic kidney cells HEPES=4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HVA=high voltage-activated calcium current IgG=immunoglobulin G LEMS=Lambert-Eaton Myasthenic Syndrome LVA=low voltage-activated calcium current MG=myasthenia gravis SCLC=small cell lung carcinoma, also called oat cell carcinoma VGCCs=voltage-gated Ca2+ channels
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LEMS IgG and Cav channel subunits, Page 25 Pharmacol 2002; 62: 485—496. 51. Arikanth J, Campbell KP. Auxiliary subunits: essential components of the voltage gated calcium channel complex. Curr Opin Neurobiol 2003; 13: 298—307. 52. Oh SJ, Sher E. MG and LEMS overlap syndrome: case report with electrophysiological and immunological evidence. Clin Neurophysiol 2005; 116: 1167—1171. 53. Roohi F, Smith PR, Bergman M, Baig MA, Sclar G. A diagnostic and management dilemma: combined paraneoplastic myasthenia gravis and Lambert-Eaton myasthenic syndrome presenting as acute respiratory failure. Neurologist 2006; 12: 322—326. 54. Kim JA, Lim YM, Jang EH, Kim KK. A patient with coexisting myasthenia gravis and lambert-eaton myasthenic syndrome. J Clin Neurol 2012; 8: 235—237. 55. Sha SJ, Layzer RB. Myasthenia gravis and Lambert-Eaton myasthenic syndrome in the same patient. Muscle Nerve 2007; 36: 115—117.
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HEK293 cells transfected with specific α1 subunit show positive reactivity to their specific antibodies. In all figures, the left panel shows GFP fluorescence as a transfection reporter. In the right panels, the same microscopic field is shown with fluorescence from the label on secondary antibodies. No cross-reactivity was ever seen with a different subunit antibody (results not presented). 176x231mm (300 x 300 DPI)
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HEK293 cells transfected with full complement of Ca2+ channel subunits (α1, β3 and α2δ) show reactivity to LEMS patient immunoglobulins. Results are from one of the three patients tested, all positive. Because the GFP reporter cDNA was used at approximately 1/5 the amount of Ca2+ channel cDNA, there are many more transfected cells with positive secondary antibody fluorescence than with GFP fluorescence. 165x181mm (300 x 300 DPI)
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Cells transfected with only the β3 subunit demonstrated binding of all LEMS and SCLC patients but not MG patient. 167x253mm (300 x 300 DPI)
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LEMS patient IgG showed reactivity to cells transfected with only a single type of α1 subunit (α1A, α1B-1, α1C-1, or α1E). Although data are presented from only one patient, identical results were seen with all three LEMS patients IgG tested. 153x182mm (300 x 300 DPI)
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