[Dosage and specificity of anti-calcium channel antibodies in Lambert-Eaton myasthenic syndrome]. / Dosage et spécificité d'autoanticorps anti-canaux calcium dans le syndrome myasthénique de Lambert-Eaton.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune channelopathy in which patients produce autoantibodies directed against voltage-gated calcium channels. Autoantibodies down-regulate calcium channels resulting in reduced transmitter release, which in turn leads to muscular weakness and autonomic dysfunction. LEMS is paraneoplastic in 60-70% of patients, most frequently associated with small cell lung carcinoma (SCLC). SCLC lines express many neuronal and neuroendocrine proteins including neuronal calcium channels of the Cav2 family (P/Q and N-type channels). It is thus likely that the paraneoplastic form of LEMS is the consequence of an anti-tumoral immune response and the production of antibodies that cross-react with identical or homologous antigens in nerve terminals. Neurological symptoms generally appear several Months before detection of the tumor. Consequently correct diagnosis of LEMS is crucial as it can allow early treatment of a particularly aggressive carcinoma. Based on published studies, our laboratory has set-up serological assays for LEMS autoantibodies as an aid to diagnosis. Calcium channels in detergent extracts of rat brain or cerebellum membranes were labeled with radioligands specific for N-type (125I-omega conotoxin GVIA) or P/Q-type (125I-omega conotoxin MVIIC) calcium channels. Autoantibodies that immunoprecipitate the ligand/channel complex can thus be titrated. Analysis of 31 LEMS sera revealed the presence of anti-N type channel antibodies in 58% and anti-P/Q type channel antibodies in 74% of patients with titres ranging from 90 to 2950 pM. Only 5 patients were seronegative in both tests, thus a combination of the two assays reliably detected autoantibodies in 26/31 (84%) patients.

Role of Thr(11) in the binding of omega-conotoxin MVIIC to N-type Ca2+ channels.

As replacement of Thr(11) of omega-conotoxin MVIIC with Ala significantly reduced the affinity for both N- and P/Q-type calcium channels, we examined the effect of substitution at this position with other residues. Binding assays using rat cerebellar P2 membranes showed that the affinity is in the order of Leu>Val, aminobutyric acid, Thr>Asn&z.Gt;Ser, Ala, Asp, Phe, Tyr for N-type channels and Thr>Leu, Val, aminobutyric acid, Asn, Ser>Ala&z.Gt;Asp, Phe, Tyr for P/Q-type channels, suggesting that aliphatic amino acids with longer side chains are favorable for block of N-type channels. The effects of substitution were examined electrophysiologically in BHK cells expressing N-type Ca2+ channels. Inhibition of Ba2+ current by the analogs did not completely correlate with binding affinity, although binding to BHK cells was comparable to rat cerebellar membranes.

alpha-latrotoxin forms calcium-permeable membrane pores via interactions with latrophilin or neurexin.

In order to explore the mechanisms by which alpha-latrotoxin activates neurotransmitter release, we have characterized its effects by patch-clamp methods on cells heterologously expressing its receptors, latrophilin-1 or neurexin-Ialpha. Application of alpha-latrotoxin (1 nM) to cells expressing rat latrophilin or neurexin, but not mock-transfected cells, induced a cationic conductance. In cells expressing latrophilin, current development was slow in the absence of divalent cations, but was accelerated by Ca2+ or Mg2+. In cells expressing neurexin, alpha-latrotoxin did not elicit currents in the absence of Ca2+. The toxin-induced conductance was rectifying, persistent, permeable to monovalent and divalent cations, but blocked by La3+. Single-channel recording revealed a permanently open state, with the same unitary conductance irrespective of whether cells expressed latrophilin or neurexin. Therefore, while pore formation displayed differences consistent with the reported properties of alpha-latrotoxin binding to latrophilin and neurexin, the pores induced by alpha-latrotoxin had identical properties. These results suggest that after anchoring to either of its nerve terminal receptors, alpha-latrotoxin inserts into the membrane and constitutes a single type of transmembrane ion pore.

Humoral immunity against glutamic acid decarboxylase and tyrosine phosphatase IA-2 in Lambert-Eaton myasthenic syndrome.

Some beta-cell-specific autoantigens also are present in the central nervous system. Furthermore, stiff man syndrome, an autoimmune neurological disease, is frequently associated with diabetes and shares with this one an anti-GAD and IA-2 humoral immunoreactivity. We wondered whether these autoantibodies could be found in other neurological diseases with a present or supposed autoimmune origin. So, anti-GAD65 (GAD65A) and anti-IA-2 (IA-2A) autoantibodies were assayed in various neurological diseases. There was a higher prevalence of such antibodies in Lambert-Eaton myasthenic syndrome (LEMS) (GAD65A, 35%; IA-2A, 21%; double positivity, 18%) compared to amyotrophic lateral sclerosis (18%, 12%, and 12%, respectively) and multiple sclerosis (10%, 3%, and 3%, respectively). In LEMS, the humoral reaction was more frequent and/or appeared earlier in the paraneoplastic forms. The detection of such autoantibodies in patients with small-cell lung carcinoma (SCLC) without LEMS suggests that these autoantigens, GAD65 and IA-2, could be produced by SCLC tissue.

Binding of six chimeric analogs of omega-conotoxin MVIIA and MVIIC to N- and P/Q-type calcium channels.

Replacement of the N-terminal half of omega-conotoxin MVIIC, a peptide blocker of P/Q-type calcium channels, with that of omega-conotoxin MVIIA significantly increased the affinity for N-type calcium channels. To identify the residues essential for subtype selectivity, we examined single reverse mutations from MVIIA-type to MVIIC-type in this chimeric analog. A reverse mutation from Lys(7) to Pro(7) decreased the affinity for both P/Q- and N-type channels, whereas that from Leu(11) to Thr(11) increased the affinity for P/Q-type channels and decreased the affinity for N-type channels. The roles of these two residues were confirmed by synthesizing two MVIIC analogs in which Pro(7) and Thr(11) were replaced with Lys(7) and Leu(11), respectively.

Binding of Ala-scanning analogs of omega-conotoxin MVIIC to N- and P/Q-type calcium channels.

omega-Conotoxin MVIIC binds to P/Q-type calcium channels with high affinity and N-type channels with low affinity. To reveal the residues essential for subtype selectivity, we synthesized Ala-scanning analogs of MVIIC. Binding assays using rat cerebellar P(2) membranes suggested that Thr(11), Tyr(13) and Lys(2) are essential for binding to both N- and P/Q-type channels, whereas Lys(4) and Arg(22) are important for binding to P/Q-type channels. These results suggest that MVIIC interacts with P/Q-type channels via a large surface, in good agreement with previous observations using chimeric analogs.

Redistribution of presynaptic proteins during alpha-latrotoxin-induced release of neurotransmitter and membrane retrieval at the frog neuromuscular junction.

Calcium-dependent exocytosis at the nerve terminal involves the synaptic core (SNARE) complex composed of the t-SNAREs syntaxin 1 and synaptosome-associated protein of 25 kDa (SNAP-25), and the v-SNARE vesicle-associated membrane protein (VAMP/synaptobrevin), a stable heterotrimer which can associate with the putative calcium sensor protein, synaptotagmin. The distribution of these proteins at the frog neuromuscular junction was examined by immunofluorescent staining and confocal microscopy following exocytosis induced by alpha-latrotoxin. Experiments were performed under conditions in which synaptic vesicle recycling was either maintained in balance with exocytosis, or completely blocked, or during recovery from block of endocytosis. When endocytosis was maintained, protein distribution was essentially identical to that of unstimulated nerve terminals, in which syntaxin 1 and SNAP-25 are localized to the presynaptic active zones coincident with the postsynaptic folds that contain a high density of acetylcholine receptors (AChRs). Block of endocytosis led to complete incorporation of vesicle proteins into the plasmalemma, and t-SNARE distribution was no longer restricted to active zones. Five minutes after the onset of recovery, both synaptic vesicle proteins and t-SNARE proteins were concentrated into small spots, in a similar pattern to that obtained following endocytosis of the vital styryl dye FM1-43. These findings are consistent with a model in which following sustained exocytosis, t-SNARE trafficking involves internalization and transit via a vesicular compartment before recycling to the presynaptic plasma membrane.

Antibodies against the beta subunit of voltage-dependent calcium channels in Lambert-Eaton myasthenic syndrome.

Lambert-Eaton myasthenic syndrome is an autoimmune disease that impairs neuromuscular transmission. Several studies suggest that neurotransmitter release is reduced by an immune response directed against the calcium channel complex of nerve terminals. The immunoglobulin G fractions from Lambert-Eaton myasthenic syndrome patients immunoprecipitate solubilized neuronal N- and P/Q-type channels and in certain cases brain, skeletal and cardiac muscle L-type channels [El Far O. et al. (1995) J. Neurochem. 64, 1696-1702; Lennon V. A. and Lambert E. H. (1989) Mayo Clin. Proc. 64, 1498-1504; Sher E. et al. (1989) Lancet ii, 640-643; Suenaga A. et al. (1996) Muscle Nerve 19, 1166-1168]. These channel immunoprecipitation assays are considered as useful for the diagnosis of this syndrome. In this study, we demonstrate that two predominant neuronal voltage-dependent calcium channel beta subunits (beta3 and beta4, of mol. wt 58,000) are general targets of Lambert-Eaton myasthenic syndrome autoantibodies. Of 20 disease sera tested, 55% were able to immunoprecipitate 35S-labeled beta subunits. All five patients affected with small-cell lung carcinoma were positive for the beta-subunit immunoprecipitation assay. Interestingly, only a fraction of the beta-subunit-positive sera was also able to immunoprecipitate N- and P/Q-type channels, suggesting that several of the beta-subunit epitopes are masked in native channels. In accordance with this observation, we found that several beta-positive sera were able to prevent the interaction between calcium channel alpha1 and beta subunits in vitro. In cases where sera were able to immunoprecipitate beta subunits, N- and P/Q-type channels, the immunoprecipitation of both channel types was either partially or entirely mediated by beta-subunit antibodies. Our results suggest that assays based on the immunoprecipitation of beta subunits can be used as an additional test to assist in the diagnosis of Lambert-Eaton myasthenic syndrome.

Interactions between proteins implicated in exocytosis and voltage-gated calcium channels.

Neurotransmitter release from synaptic vesicles is triggered by voltage-gated calcium influx through P/Q-type or N-type calcium channels. Purification of N-type channels from rat brain synaptosomes initially suggested molecular interactions between calcium channels and two key proteins implicated in exocytosis: synaptotagmin I and syntaxin 1. Co-immunoprecipitation experiments were consistent with the hypothesis that both N- and P/Q-type calcium channels, but not L-type channels, are associated with the 7S complex containing syntaxin 1, SNAP-25, VAMP and synaptotagmin I or II. Immunofluorescence confocal microscopy at the frog neuromuscular junction confirmed that calcium channels, syntaxin 1 and SNAP-25 are co-localized at active zones of the presynaptic plasma membrane where transmitter release occurs. Experiments with recombinant proteins were performed to map synaptic protein interaction sites on the alpha 1A subunit, which forms the pore of the P/Q-type calcium channel. In vitro-translated 35S-synaptotagmin I bound to a site located on the cytoplasmic loop linking homologous domains II and III of the alpha 1A subunit. This direct link would target synaptotagmin, a putative calcium sensor for exocytosis, to a microdomain of calcium influx close to the channel mouth. Cysteine string proteins (CSPs) contain a J-domain characteristic of molecular chaperones that cooperate with Hsp70. They are located on synaptic vesicles and thought to be involved in modulating the activity of presynaptic calcium channels. CSPs were found to bind to the same domain of the calcium channel as synaptotagmin, and also to associate with VAMP. CSPs may act as molecular chaperones in association with Hsp70 to direct assembly or dissociation of multiprotein complexes at the calcium channel.

Binding of chimeric analogs of omega-conotoxin MVIIA and MVIIC to the N- and P/Q-type calcium channels.

Despite their high sequence homology, the peptide neurotoxins omega-conotoxin MVIIA and MVIIC selectively block N- and P/Q-type calcium channels, respectively. To study the recognition mechanism of calcium channel subtypes, two chimeric analogs of omega-conotoxin MVIIA and MVIIC were synthesized by exchanging their N- and C-terminal halves. Binding assay for both N- and P/Q-type calcium channels showed that amino acid residues restricted to the N-terminal half are important for the recognition of N-type channels, whereas essential residues for P/Q-type channel recognition are widely spread over the whole omega-conotoxin molecule.

Immunoassays fail to detect antibodies against neuronal calcium channels in amyotrophic lateral sclerosis serum.

Recent studies suggested that autoantibodies that bind to voltage-dependent calcium channels and activate calcium entry may play a role in the progressive degeneration of motoneurons in sporadic amyotrophic lateral sclerosis. Immunoassays were performed to assess autoantibody titer in patients with amyotrophic lateral sclerosis or Lambert-Eaton myasthenic syndrome, a disease in which the presence of anti-calcium channel antibodies is well documented. Based on immunoprecipitation assays for antibodies against N-type calcium channels, only 8% (2/25) of amyotrophic lateral sclerosis patients had marginally positive titers, whereas 58% (18/31) of patients with Lambert-Eaton myasthenic syndrome had positive titers. Enzyme-linked immunosorbent assays with purified neuronal N-type calcium channels revealed immunoreactivity in 2 of 25 amyotrophic lateral sclerosis sera and 12 of 31 Lambert-Eaton myasthenic syndrome sera, which is not compatible with suggestions that enzyme-linked immunosorbent assay is a more sensitive technique for the detection of autoantibodies in amyotrophic lateral sclerosis. Furthermore, based on immunoprecipitation assays, amyotrophic lateral sclerosis sera were totally negative for antibodies against L-type calcium channels from skeletal muscle or brain. These data do not support the hypothesis that an autoimmune response against calcium channels plays a primary role in amyotrophic lateral sclerosis.

Interaction of SNARE complexes with P/Q-type calcium channels in rat cerebellar synaptosomes.

P- and Q-type calcium channels, which trigger rapid neurotransmitter release at many mammalian synapses, are blocked by omega-conotoxin MVIIC. 125I-omega-Conotoxin MVIIC binding to rat cerebellar synaptosomes was not displaced by omega-conotoxins GVIA or MVIIA (Ki > 1 microM), which are selective for N-type calcium channels. Solubilized 125I-omega-conotoxin MVIIC receptors were specifically recognized by antibodies directed against alpha1A calcium channel subunits, proteins known to constitute a pore with P/Q-like channel properties. Antibodies against syntaxin 1, SNAP 25, and VAMP 2 (synaptobrevin) each immunoprecipitated a similar fraction (20-40%) of omega-conotoxin MVIIC receptors. Immunoprecipitation was not additive, suggesting that heterotrimeric (SNARE) complexes containing these three proteins interact with P/Q-type calcium channels. Immobilized monoclonal anti-syntaxin antibodies retained alpha1A calcium channel subunits of 220, 180 and 160 kDa monitored by immunoblotting with site directed antibodies. Synaptotagmin was detected in channel-associated complexes, but not synaptophysin, Rab 3A nor rat cysteine string protein. Trimeric SNARE complexes are implicated in calcium-dependent exocytosis, a process thought to be regulated by synaptotagmin. Our results indicate that these proteins interact with P/Q-type calcium channels, which may optimize their location within domains of calcium influx.

Tyr13 is essential for the binding of omega-conotoxin MVIIC to the P/Q-type calcium channel.

An analog of omega-conotoxin MVIIC (Y13A-MVIIC) was synthesized by replacing Tyr13 with Ala to study the role of Tyr13 residue conserved in many omega-conotoxins. Y13A-MVIIC has an overall conformation similar to that of the native toxin, but an enormously reduced ability to displace 125I-omega-conotoxin MVIIC binding to rat cerebellar P2 membranes. These results suggest that Tyr13 is essential for the activity of omega-conotoxins at P/Q-type calcium channels.

Properties of omega conotoxin MVIIC receptors associated with alpha 1A calcium channel subunits in rat brain.

Solubilized 125I-omega conotoxin MVIIC receptors from rat cerebellum were immunoprecipitated by antibodies directed against the calcium channel alpha 1A subunit. Anti-alpha 1A antibodies recognized a 240-220, 180 and 160 kDa proteins in immunoblots of cerebellar membranes. Disuccinimidyl suberate cross-linked 125I-omega conotoxin MVIIC to an alpha 2 delta-like 200-180 kDa subunit, which migrated at 150-140 kDa after disulfide reduction. These observations are consistent with a heteromeric structure in which high affinity omega conotoxin MVIIC binding sites formed by alpha 1A subunits are located in close proximity to peripheral alpha 2 subunits.

Antigens associated with N- and L-type calcium channels in Lambert-Eaton myasthenic syndrome.

In Lambert-Eaton myasthenic syndrome neurotransmitter release is reduced by an autoimmune response directed against the calcium channel complex of the nerve terminal. Autoantibodies were detected by immunoprecipitation assays using solubilized receptors labeled with ligands selective for N-type (125I-omega conotoxin GVIA) and L-type ([3H]PN200-110) calcium channels. Sera with a high antibody titer (> 3 nM) against rat brain N-type channels contained autoantibodies that immunoprecipitated neuronal and muscle L-type channels. These IgG fractions stained a 55-kDa protein in immunoblots of purified skeletal muscle dihydropyridine receptor, suggesting that they contain autoantibodies against the beta subunit of the calcium channel. A distinct antibody population in the same fractions reacted with a nerve terminal 65-kDa protein that is unrelated to the beta subunit and displays properties similar to those of synaptotagmin.

Binding of Lambert-Eaton myasthenic syndrome IgG to synaptosomal proteins does not correlate with an inhibition of calcium uptake.

The immunochemical and functional properties of IgG fractions from patients with Lambert-Eaton myasthenic syndrome (LEMS) were examined in chick and rat synaptosomes. LEMS IgG immunoprecipitated 125I-omega conotoxin GVIA (125I-omega CgTx) labeled N-type calcium channels solubilized from both tissues, and reacted with a 65 kDa protein band in immunoblots of rat synaptosomes. Depolarization-induced 45Ca2+ influx into chick synaptosomes was partially inhibited by omega CgTx, whereas influx into rat synaptosomes was insensitive to omega CgTx. No effect of LEMS sera or IgG on 45Ca2+ uptake was apparent in either preparation.

Interaction of a synaptobrevin (VAMP)-syntaxin complex with presynaptic calcium channels.

Nerve terminal protein complexes implicated in exocytosis were examined by immuno-isolation from rat brain synaptosomes. Immunoprecipitation with anti-syntaxin or anti-VAMP antibodies revealed a syntaxin-SNAP25-VAMP-synaptotagmin complex. Anti-VAMP antibodies also trapped a distinct VAMP-synaptophysin complex. A similar fraction (about 70%) of N-type calcium channels ([125I]omega conotoxin GVIA receptors), was immunoprecipitated by either anti-syntaxin or anti-VAMP antibodies, but not by anti-synaptophysin antibodies (< 4%). The majority of N- but not L-type calcium channels ([3H]PN200-110 receptors), appear to be associated with a synaptic vesicle prefusion complex.

Purification of the N-type calcium channel associated with syntaxin and synaptotagmin. A complex implicated in synaptic vesicle exocytosis.

omega-Conotoxin-sensitive N-type calcium channels control neurotransmitter release at the nerve terminal and interact with proteins implicated in secretion. Solubilized omega-conotoxin receptors from rat brain synaptic membrane were immunoprecipitated by antibodies against calcium channel alpha 1 subunits, syntaxin, and a 105-kDa plasma membrane protein. A multimeric complex, composed of calcium channel subunits, and synaptic proteins that showed varying degrees of association, was purified by a procedure involving anti-syntaxin immunoaffinity chromatography. A 250-kDa N-type alpha 1 subunit, containing cAMP-dependent phosphorylation site(s), was identified by photoaffinity labeling with 125I-azidonitrobenzoyl omega-conotoxin and immunoblotting with sequence-directed antibodies. An immunologically related 210-kDa form of the alpha 1 subunit was detected that displayed different pharmacological and regulatory properties. Protein bands of 140, 70, 58, and 35 kDa comigrated with purified alpha 1 subunits upon sucrose gradient centrifugation, whereas the 105-kDa protein was removed. The 58- and 35-kDa bands contained, respectively, the synaptic vesicle protein synaptotagmin and syntaxin, a plasma membrane protein that binds synaptic vesicle proteins. Purified omega-contoxin receptors were quantitatively immunoprecipitated by anti-syntaxin antibodies. These proteins may constitute an isolated exocytotic complex in which the N-type calcium channel tightly interacts with a synaptic vesicle docking site.

[Lambert-Eaton syndrome without calcium channel autoantibodies]. / Syndrome de Lambert-Eaton sans anticorps anti-canaux calciques.

Two cases of Lambert-Eaton syndrome are reported, one associated with a small cell lung carcinoma, the other without any etiology at the time of the study. None of these cases showed significant titers of calcium channel autoantibodies. The heterogeneity of the Lambert-Eaton syndrome and the responsibility of the autoantibodies detected by immunoprecipitation of the voltage-gated calcium channel in the occurrence of the neuromuscular block are discussed.