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. 2017 Apr;88(4):353-361.
doi: 10.1136/jnnp-2016-314758. Epub 2017 Jan 23.

Intracellular and Non-Neuronal Targets of Voltage-Gated Potassium Channel Complex Antibodies

Free PMC article

Intracellular and Non-Neuronal Targets of Voltage-Gated Potassium Channel Complex Antibodies

Bethan Lang et al. J Neurol Neurosurg Psychiatry. .
Free PMC article


Objectives: Autoantibodies against the extracellular domains of the voltage-gated potassium channel (VGKC) complex proteins, leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein-2 (CASPR2), are found in patients with limbic encephalitis, faciobrachial dystonic seizures, Morvan's syndrome and neuromyotonia. However, in routine testing, VGKC complex antibodies without LGI1 or CASPR2 reactivities (double-negative) are more common than LGI1 or CASPR2 specificities. Therefore, the target(s) and clinical associations of double-negative antibodies need to be determined.

Methods: Sera (n=1131) from several clinically defined cohorts were tested for IgG radioimmunoprecipitation of radioiodinated α-dendrotoxin (125I-αDTX)-labelled VGKC complexes from mammalian brain extracts. Positive samples were systematically tested for live hippocampal neuron reactivity, IgG precipitation of 125I-αDTX and 125I-αDTX-labelled Kv1 subunits, and by cell-based assays which expressed Kv1 subunits, LGI1 and CASPR2.

Results: VGKC complex antibodies were found in 162 of 1131 (14%) sera. 90 of these (56%) had antibodies targeting the extracellular domains of LGI1 or CASPR2. Of the remaining 72 double-negative sera, 10 (14%) immunoprecipitated 125I-αDTX itself, and 27 (38%) bound to solubilised co-expressed Kv1.1/1.2/1.6 subunits and/or Kv1.2 subunits alone, at levels proportionate to VGKC complex antibody levels (r=0.57, p=0.0017). The sera with LGI1 and CASPR2 antibodies immunoprecipitated neither preparation. None of the 27 Kv1-precipitating samples bound live hippocampal neurons or Kv1 extracellular domains, but 16 (59%) bound to permeabilised Kv1-expressing human embryonic kidney 293T cells. These intracellular Kv1 antibodies mainly associated with non-immune disease aetiologies, poor longitudinal clinical-serological correlations and a limited immunotherapy response.

Conclusions: Double-negative VGKC complex antibodies are often directed against cytosolic epitopes of Kv1 subunits and occasionally against non-mammalian αDTX. These antibodies should no longer be classified as neuronal-surface antibodies. They consequently lack pathogenic potential and do not in themselves support the use of immunotherapies.

Conflict of interest statement

Competing interests: AV, SRI, BL and PW are coapplicants and receive royalties on patent application WO/2010/046716 entitled ‘Neurological Autoimmune Disorders’. The patent has been licensed to Euroimmun AG for the development of assays for LGI1 and other VGKC-complex antibodies. BL and SRI had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.


Figure 1
Figure 1
Detection of VGKC complex antibodies and antibodies to dendrotoxin. (A) VGKC complex antibodies were determined from 1131 samples, including those with known VGKC complex antibodies (both with (n=84) and without (n=27) LGI1 or CASPR2 reactivities), and unselected patients with adult-onset epilepsies, infectious diseases, autonomic syndromes, LEMS, Hu, healthy smokers and HC. Samples with LGI1 antibodies (n=69), CASPR2 antibodies (n=21) and all available samples with VGKC complex antibody levels above 100 pM and unknown antigenic targets (red; n=72) were carried forward to other assays. Dotted lines represent this cut-off and the 400 pM cut-off from a previous study; (B) 10 of the 72 samples with unknown antigens immunoprecipitated substantial quantities of of 125I-αDTX alone (dotted line at 137 pM represents the mean plus three standard deviations from 20 HCs). Three serum samples from a snake handler (grey dots) also had antibodies to 125I-αDTX; (C) 125I-αDTX antibody levels correlated with their corresponding VGKC complex antibody levels (r=0.54, p=0.015, Spearman's rank correlation). 125I-αDTX, radioiodinated α-dendrotoxin; CASPR2, contactin-associated protein-2; HC, healthy controls; Hu, Hu antibodies; LEMS, Lambert-Eaton myasthenic syndrome; LGI1, leucine-rich glioma-inactivated 1; VGKC, voltage-gated potassium channel.
Figure 2
Figure 2
Kv1 antibodies target intracellular epitopes. (A) Twenty-seven of the remaining 62 patients with unknown VGKC complex antigenic targets precipitated either 125I-αDTX-labelled Kv1.1/Kv1.2/Kv1.6 co-transfected HEK cell extracts (red circles) or 125I-αDTX-labelled Kv1.2-transfected HEK cell extracts (red circles with black outline). No sera with LGI1 or CASPR2 antibodies showed positive results. HEK cells transfected with Kv1.1 alone or Kv1.6 alone did not bind 125I-αDTX in solution; (B) a commercial antibody to the extracellular domain of Kv1.1 (anti-Kv1.1e) labelled the cell surface of live HEK cells co-transfected with Kv1.1, Kv1.2 and Kv1.6 (and enhanced green fluorescent protein (EGFP)). No patient antibodies (n=175, including the 62 double-negative samples without αDTX reactivity) showed similar binding to these live cells or live cells expressing only one of these subunits; (C) binding to fixed Kv1-transfected HEK cells was seen using serum samples which precipitated Kv1s from solution. This co-localised with binding of commercial antibodies against the intracellular domain of Kv1.2 (anti-Kv1.2). Examples for Kv1.2 and Kv1.6 are shown in online supplementary figure S1C. Scale bar=10 microns. 125I-αDTX, radioiodinated α-dendrotoxin; CASPR2, contactin-associated protein-2; HEK, human embryonic kidney 293T; LGI1, leucine-rich glioma-inactivated 1; VGKC, voltage-gated potassium channel.
Figure 3
Figure 3
Summary of the sequential flow of assays through the study. As shown in figure 1A, 1131 samples were initially tested for VGKC complex antibodies by RIA (VGKC complex RIA, A) and subsequently using LGI1 and CASPR2 antibody live CBAs (B), live neuronal cultures (C) and precipitation of αDTX (αDTX RIA, D). As detailed in figure 2, double-negative samples were then tested for binding to the extracellular domains of live Kv1-tranfected HEK cells (Kv1-live CBA), for immunoprecipitations of 125I-αDTX-labelled Kv1-transfected HEK cells (Kv1-HEK RIA, E) and for binding to fixed permeabilised Kv1-transfected HEK cells (Kv1-fixed CBA). Cohorts are defined in more detail in the Methods section and online supplementary table S1. 125I-αDTX, radioiodinated α-dendrotoxin; CASPR2, contactin-associated protein-2; CBA, cell-based assay; HEK, human embryonic kidney 293T; Hu, Hu antibodies; LEMS, Lambert-Eaton myasthenic syndrome; LGI1, leucine-rich glioma-inactivated 1; RIA, radioimmunoassay; VGKC, voltage-gated potassium channel.
Figure 4
Figure 4
Molecular and clinical features associated with double-negative VGKC complex antibodies. (A) The illustration of study results demonstrates that the antibodies with pathogenic potential (blue and green) target the extracellular domains of LGI1 and CASPR2, respectively, whereas likely non-pathogenic antibodies (red) target the intracellular domain of Kv1 channels, especially Kv1.2, and the α-dendrotoxin molecule itself (yellow), which is not present in mammalian tissue. Other intracellular targets may include the Kv-β2 subunit (pink). (B) The patients with intracellular Kv1 antibodies had no clear peak age of onset, and (C) 12 showed varied, known diagnoses (*), and 15 had conditions of unknown aetiology, unlikely to be autoimmune. CASPR2, contactin-associated protein-2; HS, hippocampal sclerosis; LGI1, leucine-rich glioma-inactivated 1; VGKC, voltage-gated potassium channel.

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