The downregulation of Kv1 channels in Lgi1-/-mice is accompanied by a profound modification of its interactome and a parallel decrease in Kv2 channels.

In animal models of LGI1-dependent autosomal dominant lateral temporal lobe epilepsy, Kv1 channels are downregulated, suggesting their crucial involvement in epileptogenesis. The molecular basis of Kv1 channel-downregulation in LGI1 knock-out mice has not been elucidated and how the absence of this extracellular protein induces an important modification in the expression of Kv1 remains unknown. In this study we analyse by immunofluorescence the modifications in neuronal Kv1.1 and Kv1.2 distribution throughout the hippocampal formation of LGI1 knock-out mice. We show that Kv1 downregulation is not restricted to the axonal compartment, but also takes place in the somatodendritic region and is accompanied by a drastic decrease in Kv2 expression levels. Moreover, we find that the downregulation of these Kv channels is associated with a marked increase in bursting patterns. Finally, mass spectrometry uncovered key modifications in the Kv1 interactome that highlight the epileptogenic implication of Kv1 downregulation in LGI1 knock-out animals.

Patient-derived antibodies reveal the subcellular distribution and heterogeneous interactome of LGI1.

Autoantibodies against leucine-rich glioma-inactivated 1 (LGI1) occur in patients with encephalitis who present with frequent focal seizures and a pattern of amnesia consistent with focal hippocampal damage. To investigate whether the cellular and subcellular distribution of LGI1 may explain the localization of these features, and hence gain broader insights into LGI1's neurobiology, we analysed the detailed localization of LGI1 and the diversity of its protein interactome, in mouse brains using patient-derived recombinant monoclonal LGI1 antibodies. Combined immunofluorescence and mass spectrometry analyses showed that LGI1 is enriched in excitatory and inhibitory synaptic contact sites, most densely within CA3 regions of the hippocampus. LGI1 is secreted in both neuronal somatodendritic and axonal compartments, and occurs in oligodendrocytic, neuro-oligodendrocytic and astro-microglial protein complexes. Proteomic data support the presence of LGI1-Kv1-MAGUK complexes, but did not reveal LGI1 complexes with postsynaptic glutamate receptors. Our results extend our understanding of regional, cellular and subcellular LGI1 expression profiles and reveal novel LGI1-associated complexes, thus providing insights into the complex biology of LGI1 and its relationship to seizures and memory loss.

Gangliosides interact with synaptotagmin to form the high-affinity receptor complex for botulinum neurotoxin B.

Botulinum neurotoxin type B (BoNT/B) recognizes nerve terminals by binding to 2 receptor components: a polysialoganglioside, predominantly GT1b, and synaptotagmin 1/2. It is widely thought that BoNT/B initially binds to GT1b then diffuses in the plane of the membrane to interact with synaptotagmin. We have addressed the hypothesis that a GT1b-synaptotagmin cis complex forms the BoNT/B receptor. We identified a consensus glycosphingolipid-binding motif in the extracellular juxtamembrane domain of synaptotagmins 1/2 and confirmed by Langmuir monolayer, surface plasmon resonance, and circular dichroism that GT1b interacts with synaptotagmin peptides containing this sequence, inducing α-helical structure. Molecular modeling and tryptophan fluorescence spectroscopy were consistent with the intertwining of GT1b and synaptotagmin, involving cis interactions between the oligosaccharide and ceramide moieties of GT1b and the juxtamembrane and transmembrane domains of synaptotagmin, respectively. Furthermore, a point mutation on synaptotagmin, located outside of the BoNT/B-binding segment, inhibited GT1b binding and blocked GT1b-induced potentiation of BoNT/B binding to synaptotagmin-expressing cells. Our findings are consistent with a model in which a preassembled GT1b-synaptotagmin complex constitutes the high-affinity BoNT/B receptor.