A causal role for TRESK loss of function in migraine mechanisms.

The two-pore potassium channel, TRESK has been implicated in nociception and pain disorders. We have for the first time investigated TRESK function in human nociceptive neurons using induced pluripotent stem cell-based models. Nociceptors from migraine patients with the F139WfsX2 mutation show loss of functional TRESK at the membrane, with a corresponding significant increase in neuronal excitability. Furthermore, using CRISPR-Cas9 engineering to correct the F139WfsX2 mutation, we show a reversal of the heightened neuronal excitability, linking the phenotype to the mutation. In contrast we find no change in excitability in induced pluripotent stem cell derived nociceptors with the C110R mutation and preserved TRESK current; thereby confirming that only the frameshift mutation is associated with loss of function and a migraine relevant cellular phenotype. We then demonstrate the importance of TRESK to pain states by showing that the TRESK activator, cloxyquin, can reduce the spontaneous firing of nociceptors in an in vitro human pain model. Using the chronic nitroglycerine rodent migraine model, we demonstrate that mice lacking TRESK develop exaggerated nitroglycerine-induced mechanical and thermal hyperalgesia, and furthermore, show that cloxyquin conversely is able to prevent sensitization. Collectively, our findings provide evidence for a role of TRESK in migraine pathogenesis and its suitability as a therapeutic target.

The Role of TRESK in Discrete Sensory Neuron Populations and Somatosensory Processing.

Two-pore domain K+ (K2P) channels generate K+ leak current, which serves a vital role in controlling and modulating neuronal excitability. This diverse family of K+ channels exhibit distinct expression and function across neuronal tissues. TWIK-related spinal cord K+ channel (TRESK) is a K2P channel with a particularly enriched role in sensory neurons and in vivo pain pathways. Here, we explored the role of TRESK across molecularly distinct sensory neuron populations and assessed its contribution to different sensory modalities. We found TRESK mRNA only in select populations of C- and A-δ nociceptors, in addition to low threshold D-hair afferents. Neurons from mice in which TRESK has been ablated demonstrated marked hyperexcitability, which was amplified under inflammatory challenge. Detailed behavioral phenotyping of TRESK knockout mice revealed specific deficits in somatosensory processing of noxious and non-noxious stimuli. These results demonstrate novel roles of TRESK in somatosensory processing and offer important information to those wishing to target the channel for therapeutic means.

Association of Leucine-Rich Glioma Inactivated Protein 1, Contactin-Associated Protein 2, and Contactin 2 Antibodies With Clinical Features and Patient-Reported Pain in Acquired Neuromyotonia.

Importance: Although acquired autoimmune neuromyotonia (NMT) is associated with voltage-gated potassium channel (VGKC)-complex antibodies, to date there has been no systematic study of autoantibodies to the specific antigens leucine-rich glioma inactivated protein 1 (LGI1), contactin-associated protein 2 (CASPR2), and contactin 2 together with the full clinical syndrome, particularly pain and autonomic and central nervous system involvement. Objectives: To study the full spectrum of clinical features and serum autoantibodies in patients with NMT, including the effects of pain on quality of life. Design, Setting, and Participants: A cohort study of clinical features and serologic testing in 38 patients with electrophysiologically-confirmed NMT, reviewed clinically between February 2007 and August 2009, in the Universities of Sydney and Kagoshima and followed up across 2 to 4 years. Association of NMT with quality of life was researched in an independent, patient-led, online pain survey conducted from April 2012 to May 2012. Serologic analyses were performed in 2012, and final data analysis was performed in 2016. Main Outcomes and Measures: Clinical data and scores on the modified Rankin Scale (mRS), which measures disability on a range of 0 to 6, with 0 indicating normal and 6 indicating death, before and after treatments were combined with CASPR2, LGI1, and contactin 2 antibody status. Results: Among the 38-person NMT cohort, 25 (65.8%) were male and the median (range) age was 55 (12-85) years. Twenty-three (60.5%) were Japanese and 15 (39.5%) were of white race/ethnicity. Symptomatic treatments (mainly antiepileptic drugs) were used in most patients with mild disease (12 patients with mRS <3), whereas immunotherapies were successful in most patients with mRS scores greater than 2. Autoantibodies to VGKC-complex antigens (17 patients [45%]), bound to CASPR2 (5 [13%]), contactin 2 (5 patients, 1 with CASPR2 [13%]), LGI1 (2 [5%]), or both LGI1 and CASPR2 (6 [16%]). The last group of 6 patients had high mRS scores (mean [SD], 3.8 [1.7]), thymoma (4 patients), pain (5 patients), autonomic (6 patients) and sleep (5 patients) disturbance, suggesting Morvan syndrome. The 56 responders to the independent patient-led survey reported pain that could be severe, anatomically widespread, and that often resulted in unemployment, domestic problems, and poor quality of life. Conclusions and Relevance: The cohort study detailed underrecognized aspects of the clinical and serologic spectrum of NMT. The heterogeneity of clinical features and of specific antibodies limit associations, but the common existence of thymoma, pain, and autonomic and central nervous system features, often with both LGI1 and CASPR2 antibodies, should be better recognized to more completely address the range of comorbidities and consequences of the disease regarding quality of life.

Immune or Genetic-Mediated Disruption of CASPR2 Causes Pain Hypersensitivity Due to Enhanced Primary Afferent Excitability.

Human autoantibodies to contactin-associated protein-like 2 (CASPR2) are often associated with neuropathic pain, and CASPR2 mutations have been linked to autism spectrum disorders, in which sensory dysfunction is increasingly recognized. Human CASPR2 autoantibodies, when injected into mice, were peripherally restricted and resulted in mechanical pain-related hypersensitivity in the absence of neural injury. We therefore investigated the mechanism by which CASPR2 modulates nociceptive function. Mice lacking CASPR2 (Cntnap2-/-) demonstrated enhanced pain-related hypersensitivity to noxious mechanical stimuli, heat, and algogens. Both primary afferent excitability and subsequent nociceptive transmission within the dorsal horn were increased in Cntnap2-/- mice. Either immune or genetic-mediated ablation of CASPR2 enhanced the excitability of DRG neurons in a cell-autonomous fashion through regulation of Kv1 channel expression at the soma membrane. This is the first example of passive transfer of an autoimmune peripheral neuropathic pain disorder and demonstrates that CASPR2 has a key role in regulating cell-intrinsic dorsal root ganglion (DRG) neuron excitability.

Pathogenic potential of antibodies to the GABAB receptor.

GABAB receptor (GABABR) autoantibodies have been detected in the serum of immunotherapy-responsive patients with autoimmune encephalitis. This study aimed to investigate the effect of immunoglobulin G (IgG) from a patient with GABABR antibodies on primary neuronal cultures and acute slices of entorhinal cortex. Primary hippocampal neuronal cultures were incubated with serum immunoglobulin from patients with GABABR or AMPA receptor (AMPAR) antibodies for up to 72 h to investigate their effect on receptor surface expression. Whole-cell patch-clamp recordings from layer III pyramidal cells of the medial entorhinal cortex were used to examine the effect on neuronal activity. GABABR surface expression was unaltered by incubation with GABABR antibodies. By contrast, after 24 h application of AMPAR antibodies, AMPARs were undetectable. However, acute application of GABABR IgG decreased both the duration of network UP states and the spike rate of pyramidal cells in the entorhinal cortex. GABABR antibodies do not appear to affect GABABRs by internalization but rather reduce excitability on the medial temporal lobe networks. This unusual mechanism of action may be exploited in rational drug development strategies.

Detection methods for neural autoantibodies.

Antibodies are part of the adaptive immune response that provides protection against microorganisms. In rare instances individuals can develop antibodies that bind to normal central nervous system structures. These antibodies have been classified into two groups depending on the subcellular location of their target antigens. Biomarker antibodies bind to cytosolic or nuclear targets. They do not impact on the normal function of the cell, but are most often paraneoplastic biomarkers that may suggest screening for different cancers. The second, more recently discovered group of antibodies recognize the three-dimensional structure of native proteins that are accessible on the cell surface. Understanding of this important difference is reflected in the nature of assays used to detect antibodies in these two groups. Western blots and, more recently, line blots, both of which use linear, denatured targets, are used to detect antibodies to intracellular targets. Newer assays have been developed that maintain the native structure of protein targets to permit detection of antibodies that recognize extracellular targets. In this chapter we describe the methods used to detect both antibody types, and discuss assay cut-offs, sample handling, and which biologic fluid to test.

Antibodies to AMPA receptors in Rasmussen's encephalitis.

Rasmussen's encephalitis is a rare progressive childhood disorder characterized by frequent severe seizures, hemiparesis, encephalitis and mental deterioration, and associated with severe unilateral neuroinflammation. Autoantibodies, particularly to the GluA3 subtype of the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propinonic acid receptor (AMPAR) were reported in the 1990s but not always confirmed. To explore further, sera from 52 patients with Rasmussen's encephalitis were tested by cell-based assays for antibodies to AMPAR GluA1/2/3, N-methyl-d-aspartate (NMDA NR1/2b), γ-aminobutyric acid type A and B (GABAAR α1/γ2/ß2 and GABABR b1/b2) receptors, for potassium channel complex proteins, and for binding to live cortical and hippocampal neuronal cultures. Two patients' sera (3.8%) bound to HEK cells co-transfected with the GluA2 and GluA3 subunits. One additional patient had a low level of VGKC-complex antibodies. These three, and seven additional, sera bound to hippocampal cultures. No other antibodies were detected. Thus, despite the rarity of GluA2/3 antibodies, 10 patients (19.2%) had evidence of antibodies to neuronal antigens. Whether these antibodies play a primary role in RE, or appear secondary to the neuro-inflammatory damage in this highly destructive disease, requires further study.

Antibodies to GABAA receptor α1 and γ2 subunits: clinical and serologic characterization.

OBJECTIVE: To search for antibodies against neuronal cell surface proteins. METHODS: Using immunoprecipitation from neuronal cultures and tandem mass spectrometry, we identified antibodies against the α1 subunit of the γ-aminobutyric acid A receptor (GABAAR) in a patient whose immunoglobulin G (IgG) antibodies bound to hippocampal neurons. We searched 2,548 sera for antibodies binding to GABAAR α, ß, and γ subunits on live HEK293 cells and identified the class, subclass, and GABAAR subunit specificities of the positive samples. RESULTS: GABAAR-Abs were identified in 40 of 2,046 (2%) referred sera previously found negative for neuronal antibodies, in 5/502 (1%) previously positive for other neuronal surface antibodies, but not in 92 healthy individuals. The antibodies in 40% bound to either the α1 (9/45, 20%) or the γ2 subunits (9/45, 20%) and were of IgG1 (94%) or IgG3 (6%) subclass. The remaining 60% had lower antibody titers (p = 0.0005), which were mainly immunoglobulin M (IgM) (p = 0.0025), and showed no defined subunit specificity. Incubation of primary hippocampal neurons with GABAAR IgG1 sera reduced surface GABAAR membrane expression. The clinical features of 15 patients (GABAAR α1 n = 6, γ2 n = 5, undefined n = 4) included seizures (47%), memory impairment (47%), hallucinations (33%), or anxiety (20%). Most patients had not been given immunotherapies, but one with new-onset treatment-resistant catatonia made substantial improvement after plasma exchange. CONCLUSIONS: The GABAAR α1 and γ2 are new targets for antibodies in autoimmune neurologic disease. The full spectrum of clinical features, treatment responses, correlation with antibody specificity, and in particular the role of the IgM antibodies will need to be assessed in future studies.

Glycine receptor antibodies in PERM and related syndromes: characteristics, clinical features and outcomes.

The clinical associations of glycine receptor antibodies have not yet been described fully. We identified prospectively 52 antibody-positive patients and collated their clinical features, investigations and immunotherapy responses. Serum glycine receptor antibody endpoint titres ranged from 1:20 to 1:60 000. In 11 paired samples, serum levels were higher than (n = 10) or equal to (n = 1) cerebrospinal fluid levels; there was intrathecal synthesis of glycine receptor antibodies in each of the six pairs available for detailed study. Four patients also had high glutamic acid decarboxylase antibodies (>1000 U/ml), and one had high voltage-gated potassium channel-complex antibody (2442 pM). Seven patients with very low titres (<1:50) and unknown or alternative diagnoses were excluded from further study. Three of the remaining 45 patients had newly-identified thymomas and one had a lymphoma. Thirty-three patients were classified as progressive encephalomyelitis with rigidity and myoclonus, and two as stiff person syndrome; five had a limbic encephalitis or epileptic encephalopathy, two had brainstem features mainly, two had demyelinating optic neuropathies and one had an unclear diagnosis. Four patients (9%) died during the acute disease, but most showed marked improvement with immunotherapies. At most recent follow-up, (2-7 years, median 3 years, since first antibody detection), the median modified Rankin scale scores (excluding the four deaths) decreased from 5 at maximal severity to 1 (P < 0.0001), but relapses have occurred in five patients and a proportion are on reducing steroids or other maintenance immunotherapies as well as symptomatic treatments. The glycine receptor antibodies activated complement on glycine receptor-transfected human embryonic kidney cells at room temperature, and caused internalization and lysosomal degradation of the glycine receptors at 37°C. Immunoglobulin G antibodies bound to rodent spinal cord and brainstem co-localizing with monoclonal antibodies to glycine receptor-α1. Ten glycine receptor antibody positive samples were also identified in a retrospective cohort of 56 patients with stiff person syndrome and related syndromes. Glycine receptor antibodies are strongly associated with spinal and brainstem disorders, and the majority of patients have progressive encephalomyelitis with rigidity and myoclonus. The antibodies demonstrate in vitro evidence of pathogenicity and the patients respond well to immunotherapies, contrasting with earlier studies of this syndrome, which indicated a poor prognosis. The presence of glycine receptor antibodies should help to identify a disease that responds to immunotherapies, but these treatments may need to be sustained, relapses can occur and maintenance immunosuppression may be required.

Faciobrachial dystonic seizures: the influence of immunotherapy on seizure control and prevention of cognitive impairment in a broadening phenotype.

Voltage-gated potassium channel complex antibodies, particularly those directed against leucine-rich glioma inactivated 1, are associated with a common form of limbic encephalitis that presents with cognitive impairment and seizures. Faciobrachial dystonic seizures have recently been reported as immunotherapy-responsive, brief, frequent events that often predate the cognitive impairment associated with this limbic encephalitis. However, these observations were made from a retrospective study without serial cognitive assessments. Here, we undertook the first prospective study of faciobrachial dystonic seizures with serial assessments of seizure frequencies, cognition and antibodies in 10 cases identified over 20 months. We hypothesized that (i) faciobrachial dystonic seizures would show a differential response to anti-epileptic drugs and immunotherapy; and that (ii) effective treatment of faciobrachial dystonic seizures would accelerate recovery and prevent the development of cognitive impairment. The 10 cases expand both the known age at onset (28 to 92 years, median 68) and clinical features, with events of longer duration, simultaneously bilateral events, prominent automatisms, sensory aura, and post-ictal fear and speech arrest. Ictal epileptiform electroencephalographic changes were present in three cases. All 10 cases were positive for voltage-gated potassium channel-complex antibodies (346-4515 pM): nine showed specificity for leucine-rich glioma inactivated 1. Seven cases had normal clinical magnetic resonance imaging, and the cerebrospinal fluid examination was unremarkable in all seven tested. Faciobrachial dystonic seizures were controlled more effectively with immunotherapy than anti-epileptic drugs (P = 0.006). Strikingly, in the nine cases who remained anti-epileptic drug refractory for a median of 30 days (range 11-200), the addition of corticosteroids was associated with cessation of faciobrachial dystonic seizures within 1 week in three and within 2 months in six cases. Voltage-gated potassium channel-complex antibodies persisted in the four cases with relapses of faciobrachial dystonic seizures during corticosteroid withdrawal. Time to recovery of baseline function was positively correlated with time to immunotherapy (r = 0.74; P = 0.03) but not time to anti-epileptic drug administration (r = 0.55; P = 0.10). Of 10 cases, the eight cases who received anti-epileptic drugs (n = 3) or no treatment (n = 5) all developed cognitive impairment. By contrast, the two who did not develop cognitive impairment received immunotherapy to treat their faciobrachial dystonic seizures (P = 0.02). In eight cases without clinical magnetic resonance imaging evidence of hippocampal signal change, cross-sectional volumetric magnetic resonance imaging post-recovery, after accounting for age and head size, revealed cases (n = 8) had smaller brain volumes than healthy controls (n = 13) (P < 0.001). In conclusion, faciobrachial dystonic seizures can be prospectively identified as a form of epilepsy with an expanding phenotype. Immunotherapy is associated with excellent control of the frequently anti-epileptic drug refractory seizures, hastens time to recovery, and may prevent the subsequent development of cognitive impairment observed in this study.

Immunization against GAD induces antibody binding to GAD-independent antigens and brainstem GABAergic neuronal loss.

Stiff person syndrome (SPS) is a highly-disabling neurological disorder of the CNS characterized by progressive muscular rigidity and spasms. In approximately 60-80% of patients there are autoantibodies to glutamic acid decarboxylase (GAD), the enzyme that synthesizes gamma-amino butyric acid (GABA), the predominant inhibitory neurotransmitter of the CNS. Although GAD is intracellular, it is thought that autoimmunity to GAD65 may play a role in the development of SPS. To test this hypothesis, we immunized mice, that expressed enhanced green fluorescent protein (EGFP) under the GAD65 promoter, with either GAD65 (n = 13) or phosphate buffered saline (PBS) (n = 13). Immunization with GAD65 resulted in autoantibodies that immunoprecipitated GAD, bound to CNS tissue in a highly characteristic pattern, and surprisingly bound not only to GAD intracellularly but also to the surface of cerebellar neurons in culture. Moreover, immunization resulted in immunoglobulin diffusion into the brainstem, and a partial loss of GAD-EGFP expressing cells in the brainstem. Although immunization with GAD65 did not produce any behavioral abnormality in the mice, the induction of neuronal-surface antibodies and the trend towards loss of GABAergic neurons in the brainstem, supports a role for humoral autoimmunity in the pathogenesis of SPS and suggests that the mechanisms may involve spread to antigens expressed on the surface of these neurons.

Morvan syndrome: clinical and serological observations in 29 cases.

OBJECTIVE: A study was undertaken to describe the clinical spectrum, voltage-gated potassium channel (VGKC) complex antibody specificities, and central nervous system localization of antibody binding in 29 patients diagnosed with Morvan syndrome (MoS). METHODS: Clinical data were collected using questionnaires. Radioimmunoassay, cell-based assays, and mouse brain immunohistochemistry were used to characterize the serum antibodies. RESULTS: Neuromyotonia (100%), neuropsychiatric features (insomnia 89.7%, confusion 65.5%, amnesia 55.6%, hallucinations 51.9%), dysautonomia (hyperhidrosis 86.2%, cardiovascular 48.3%), and neuropathic pain (62.1%) were the most common manifestations. A total of 93.1% of MoS patients were male. VGKC-complex antibodies were present in 23 of 29 (79%) MoS patients at referral; 24 of 27 available sera had CASPR2, LGI1, or both CASPR2 and LGI1 antibodies (3 also with contactin-2 antibodies). CASPR2 antibodies were generally higher titer than LGI1 antibodies. Tumors (41.4%), mainly thymomas, were associated with CASPR2 antibodies and a poor prognosis, whereas LGI1 antibodies were associated with serum hyponatremia. In brain tissue regions including the hypothalamus, raphe, and locus coeruleus, commercial antibodies to LGI1 bound to neuronal cell bodies including the antidiuretic hormone-secreting and orexin-secreting hypothalamic neurons, whereas CASPR2 commercial antibodies bound more often to the neuropil. MoS antibodies bound similarly, but there was evidence of additional antibodies in some sera that were not adsorbed by LGI1- or CASPR2-expressing cells and bound to mouse Caspr2(-/-) tissue. INTERPRETATION: MoS is clinically distinct from other VGKC-complex antibody-associated conditions, and usually is associated with high-titer CASPR2 antibodies, often accompanied by lower-titer LGI1 antibodies. CASPR2 and LGI1 antibodies bind to multiple brain regions, which helps to explain the multifocal clinical features of this disease, but other antibodies are likely to play a role in some patients and need to be characterized in future studies.

Morvan's syndrome associated with antibodies to multiple components of the voltage-gated potassium channel complex.

We describe a patient presenting with a combination of muscle fasciculations, paresthesias, hyperhidrosis, as well as insomnia, agitation and confusion. He went on to develop psychosis and respiratory failure requiring intensive care. Electromyography confirmed the presence of neuromyotonia and CSF showed mild pleocytosis. Routine testing for voltage-gated potassium channel complex (VGKC-complex) antibodies was highly positive, confirming the clinical diagnosis of Morvan's syndrome. The patient improved after treatment with intravenous immunoglobulin and methylprednisolone. Further investigation of the antigenic targets using immunohistochemistry and cell-based assays revealed that he had autoantibodies targeting Lgi1, Caspr2 and Contactin-2/Tag-1, all proteins known to be complexed with VGKC in peripheral nerves and CNS. This is the first case of Morvan's syndrome from Cyprus and illustrates the clinical features as well as the emerging complexity of antigenic targets involved in the pathogenesis.

Faciobrachial dystonic seizures precede Lgi1 antibody limbic encephalitis.

OBJECTIVE: To describe a distinctive seizure semiology that closely associates with voltage-gated potassium channel (VGKC)-complex/Lgi1 antibodies and commonly precedes the onset of limbic encephalitis (LE). METHODS: Twenty-nine patients were identified by the authors (n = 15) or referring clinicians (n = 14). The temporal progression of clinical features and serum sodium, brain magnetic resonance imaging (MRI), positron emission tomography/single photon emission computed tomography, and VGKC-complex antibodies was studied. RESULTS: Videos and still images showed a distinctive adult-onset, frequent, brief dystonic seizure semiology that predominantly affected the arm and ipsilateral face. We have termed these faciobrachial dystonic seizures (FBDS). All patients tested during their illness had antibodies to VGKC complexes; the specific antigenic target was Lgi1 in 89%. Whereas 3 patients never developed LE, 20 of the remaining 26 (77%) experienced FBDS prior to the development of the amnesia and confusion that characterize LE. During the prodrome of FBDS alone, patients had normal sodium and brain MRIs, but electroencephalography demonstrated ictal epileptiform activity in 7 patients (24%). Following development of LE, the patients often developed other seizure semiologies, including typical mesial temporal lobe seizures. At this stage, investigations commonly showed hyponatremia and MRI hippocampal high T2 signal; functional brain imaging showed evidence of basal ganglia involvement in 5/8. Antiepileptic drugs (AEDs) were generally ineffective and in 41% were associated with cutaneous reactions that were often severe. By contrast, immunotherapies produced a clear, and often dramatic, reduction in FBDS frequency. INTERPRETATION: Recognition of FBDS should prompt testing for VGKC-complex/Lgi1 antibodies. AEDs often produce adverse effects; treatment with immunotherapies may prevent the development of LE with its potential for cerebral atrophy and cognitive impairment.

Human limbic encephalitis serum enhances hippocampal mossy fiber-CA3 pyramidal cell synaptic transmission.

PURPOSE: Limbic encephalitis (LE) is a central nervous system (CNS) disease characterized by subacute onset of memory loss and epileptic seizures. A well-recognized form of LE is associated with voltage-gated potassium channel complex antibodies (VGKC-Abs) in the patients' sera. We aimed to test the hypothesis that purified immunoglobulin G (IgG) from a VGKC-Ab LE serum would excite hippocampal CA3 pyramidal cells by reducing VGKC function at mossy-fiber (MF)-CA3 pyramidal cell synapses. METHODS: We compared the effects of LE and healthy control IgG by whole-cell patch-clamp and extracellular recordings from CA3 pyramidal cells of rat hippocampal acute slices. RESULTS: We found that the LE IgG induced epileptiform activity at a population level, since synaptic stimulation elicited multiple population spikes extracellularly recorded in the CA3 area. Moreover, the LE IgG increased the rate of tonic firing and strengthened the MF-evoked synaptic responses. The synaptic failure of evoked excitatory postsynaptic currents (EPSCs) was significantly lower in the presence of the LE IgG compared to the control IgG. This suggests that the LE IgG increased the release probability on MF-CA3 pyramidal cell synapses compared to the control IgG. Interestingly, α-dendrotoxin (120 nm), a selective Kv1.1, 1.2, and 1.6 subunit antagonist of VGKC, mimicked the LE IgG-mediated effects. CONCLUSIONS: This is the first functional demonstration that LE IgGs reduce VGKC function at CNS synapses and increase cell excitability.

Antibodies to Kv1 potassium channel-complex proteins leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 in limbic encephalitis, Morvan's syndrome and acquired neuromyotonia.

Antibodies that immunoprecipitate (125)I-alpha-dendrotoxin-labelled voltage-gated potassium channels extracted from mammalian brain tissue have been identified in patients with neuromyotonia, Morvan's syndrome, limbic encephalitis and a few cases of adult-onset epilepsy. These conditions often improve following immunomodulatory therapies. However, the proportions of the different syndromes, the numbers with associated tumours and the relationships with potassium channel subunit antibody specificities have been unclear. We documented the clinical phenotype and tumour associations in 96 potassium channel antibody positive patients (titres >400 pM). Five had thymomas and one had an endometrial adenocarcinoma. To define the antibody specificities, we looked for binding of serum antibodies and their effects on potassium channel currents using human embryonic kidney cells expressing the potassium channel subunits. Surprisingly, only three of the patients had antibodies directed against the potassium channel subunits. By contrast, we found antibodies to three proteins that are complexed with (125)I-alpha-dendrotoxin-labelled potassium channels in brain extracts: (i) contactin-associated protein-2 that is localized at the juxtaparanodes in myelinated axons; (ii) leucine-rich, glioma inactivated 1 protein that is most strongly expressed in the hippocampus; and (iii) Tag-1/contactin-2 that associates with contactin-associated protein-2. Antibodies to Kv1 subunits were found in three sera, to contactin-associated protein-2 in 19 sera, to leucine-rich, glioma inactivated 1 protein in 55 sera and to contactin-2 in five sera, four of which were also positive for the other antibodies. The remaining 18 sera were negative for potassium channel subunits and associated proteins by the methods employed. Of the 19 patients with contactin-associated protein-antibody-2, 10 had neuromyotonia or Morvan's syndrome, compared with only 3 of the 55 leucine-rich, glioma inactivated 1 protein-antibody positive patients (P < 0.0001), who predominantly had limbic encephalitis. The responses to immunomodulatory therapies, defined by changes in modified Rankin scores, were good except in the patients with tumours, who all had contactin-associated-2 protein antibodies. This study confirms that the majority of patients with high potassium channel antibodies have limbic encephalitis without tumours. The identification of leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 as the major targets of potassium channel antibodies, and their associations with different clinical features, begins to explain the diversity of these syndromes; furthermore, detection of contactin-associated protein-2 antibodies should help identify the risk of an underlying tumour and a poor prognosis in future patients.