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

Jorge Ramirez-Franco, Kévin Debreux, Marion Sangiardi, Maya Belghazi, Yujin Kim, Suk-Ho Lee, Christian Lévêque, Michael Seagar, Oussama El Far

<figure><img src="image_1.png" /></figure>

## To cite this version:

Jorge Ramirez-Franco, Kévin Debreux, Marion Sangiardi, Maya Belghazi, Yujin Kim, et al. The downregulation of Kv1 channels in Lgi1 mice is accompanied by a profound modification of its interactome and a parallel decrease in Kv2 channels. Neurobiology of Disease, 2024, 196, pp.106513. 10.1016/j.nbd.2024.106513. inserm-04572150

HAL Id: inserm-04572150

https://inserm.hal.science/inserm-04572150

Submitted on 10 May 2024

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<figure><img src="image_2.png" /></figure>

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

Jorge Ramirez-Franco, Kévin Debreux, Marion Sangiardi, Maya Belghazi, Yujin Kim, Suk-Ho Lee, Christian Lévêque, Michael Seagar, Oussama El Far

<figure><img src="image_1.png" /></figure>

## To cite this version:

Jorge Ramirez-Franco, Kévin Debreux, Marion Sangiardi, Maya Belghazi, Yujin Kim, et al. The downregulation of Kv1 channels in Lgi1 mice is accompanied by a profound modification of its interactome and a parallel decrease in Kv2 channels. Neurobiology of Disease, 2024, 196, pp.106513. 10.1016/j.nbd.2024.106513. inserm-04572150

HAL Id: inserm-04572150

https://inserm.hal.science/inserm-04572150v1

Submitted on 10 May 2024

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_3.png" /></figure>

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

Jorge Ramirez-Franco<sup>a,*</sup>, Kévin Debreux<sup>a</sup>, Marion Sangiardi<sup>a</sup>, Maya Belghazi<sup>b</sup>, Yujin Kim<sup>c</sup>, Suk-Ho Lee<sup>c</sup>, Christian Lévèque<sup>a</sup>, Michael Seagar<sup>a</sup>, Oussama El Far<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

Kv₁ channels

Kv₂ channels

Leucine-rich glioma-inactivated 1 (LGI1)

Limbic encephalitis

Autosomal-dominant lateral temporal lobe

epilepsy

Proteomic

## ABSTRACT

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.

## 1. Introduction

The delayed rectifying Kv₁ channels (KCNA, Shaker) which mediate low voltage-activated K⁺ currents are key regulators of neuronal excitability, affecting action-potential (AP) threshold and firing patterns (Johnston et al., 2010) (Jan and Jan, 2012) (Debanne et al., 2011). Functional plasticity in neurons involves dynamic changes in the expression and subcellular distribution of potassium channels in distinct neuronal compartments (Trimmer, 2015). Kv₁ channels are formed by homomeric or heteromeric co-assembly of four alpha subunits with auxiliary subunits (Trimmer, 2015). A subset of Kv₁ subunits, namely Kv₁.₁ (KCNA1), Kv₁.₂ (KCNA2) and Kv₁.₆ (KCNA6) underlie the α-dendrotoxin (α-DTX) sensitive D-type current and these Kv subunits are present in axonal and synaptic compartments (Ogawa et al., 2008) (Gu et al., 2003) as well as at axonal initial segments (Inda et al., 2006) (Seagar et al., 2017).

Gain- or loss-of-function, inherited or de novo missense variants of these subunits lead to neurological disorders which display extensive

clinical heterogeneity (Browne et al., 1994) (Herson et al., 2003) (Verdura et al., 2020) (Doring et al., 2021).

Leucine-rich glioma-inactivated 1 (LGI1) is a secreted extracellular glycoprotein (Senechal et al., 2005) which is associated with Kv₁ channels (Ogawa et al., 2010) (Schulte et al., 2006) (Irani et al., 2010). Mutations in the LGI1 gene often result in the inhibition of its secretion (Senechal et al., 2005) (Fukata et al., 2006), which underlies hyperexcitability in Autosomal Dominant Lateral Temporal Lobe Epilepsy (ADLTE). Also, the production of LGI1-autoantibodies promotes neuronal excitability and epilepsy in LGI1-dependent autoimmune limbic encephalitis. In both genetic and autoimmune disruption of LGI1 function, a significant decrease in total, as well as plasma membrane, Kv₁.₁ and Kv₁.₂ is observed (Seagar et al., 2017) (Petit-Pedrol et al., 2018) (Zhou et al., 2018). This decrease may underlie the increase in neuronal excitability in ADLTE (Seagar et al., 2017) and precedes the previously described (Fukata et al., 2021) perturbation in the AMPA/NMDA receptors ratio (Petit-Pedrol et al., 2018) (Seagar et al., 2017). An additional secretion-defective LGI1 mutation was recently found to

<footnote><sup>a</sup> INSERM UMR_S 1072, Unité de Neurobiologie des canaux Ioniques et de la Synapse, Aix-Marseille Université, 13015 Marseille, France</footnote>

<footnote><sup>b</sup> Marseille Proteomique (MaP), Plateforme Proteomique IMM, CNRS FR3479, Aix-Marseille Université, 31 Chemin Joseph Aiguier, 13009 Marseille, France</footnote>

<footnote><sup>c</sup> Department of Physiology, Cell Physiology Lab, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 03080, South Korea</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: jose-jorge.ramirez-franco@univ-amu.fr (J. Ramirez-Franco), oussama.el-far@inserm.fr (O. El Far).</footnote>
<figure><img src="image_3.png" /></figure>

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

Jorge Ramirez-Franco<sup>a,*</sup>, Kévin Debreux<sup>a</sup>, Marion Sangiardi<sup>a</sup>, Maya Belghazi<sup>b</sup>, Yujin Kim<sup>c</sup>, Suk-Ho Lee<sup>c</sup>, Christian Lévèque<sup>a</sup>, Michael Seagar<sup>a</sup>, Oussama El Far<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

Kv₁ channels

Kv₂ channels

Leucine-rich glioma-inactivated 1 (LGI1)

Limbic encephalitis

Autosomal-dominant lateral temporal lobe

epilepsy

Proteomic

## ABSTRACT

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.

## 1. Introduction

The delayed rectifying Kv₁ channels (KCNA, Shaker) which mediate low voltage-activated K⁺ currents are key regulators of neuronal excitability, affecting action-potential (AP) threshold and firing patterns (Johnston et al., 2010) (Jan and Jan, 2012) (Debanne et al., 2011). Functional plasticity in neurons involves dynamic changes in the expression and subcellular distribution of potassium channels in distinct neuronal compartments (Trimmer, 2015). Kv₁ channels are formed by homomeric or heteromeric co-assembly of four alpha subunits with auxiliary subunits (Trimmer, 2015). A subset of Kv₁ subunits, namely Kv₁.₁ (KCNA1), Kv₁.₂ (KCNA2) and Kv₁.₆ (KCNA6) underlie the α-dendrotoxin (α-DTX) sensitive D-type current and these Kv subunits are present in axonal and synaptic compartments (Ogawa et al., 2008) (Gu et al., 2003) as well as at axonal initial segments (Inda et al., 2006) (Seagar et al., 2017).

Gain- or loss-of-function, inherited or de novo missense variants of these subunits lead to neurological disorders which display extensive

clinical heterogeneity (Browne et al., 1994) (Herson et al., 2003) (Verdura et al., 2020) (Doring et al., 2021).

Leucine-rich glioma-inactivated 1 (LGI1) is a secreted extracellular glycoprotein (Senechal et al., 2005) which is associated with Kv₁ channels (Ogawa et al., 2010) (Schulte et al., 2006) (Irani et al., 2010). Mutations in the LGI1 gene often result in the inhibition of its secretion (Senechal et al., 2005) (Fukata et al., 2006), which underlies hyperexcitability in Autosomal Dominant Lateral Temporal Lobe Epilepsy (ADLTE). Also, the production of LGI1-autoantibodies promotes neuronal excitability and epilepsy in LGI1-dependent autoimmune limbic encephalitis. In both genetic and autoimmune disruption of LGI1 function, a significant decrease in total, as well as plasma membrane, Kv₁.₁ and Kv₁.₂ is observed (Seagar et al., 2017) (Petit-Pedrol et al., 2018) (Zhou et al., 2018). This decrease may underlie the increase in neuronal excitability in ADLTE (Seagar et al., 2017) and precedes the previously described (Fukata et al., 2021) perturbation in the AMPA/NMDA receptors ratio (Petit-Pedrol et al., 2018) (Seagar et al., 2017). An additional secretion-defective LGI1 mutation was recently found to

<footnote><sup>a</sup> INSERM UMR_S 1072, Unité de Neurobiologie des canaux Ioniques et de la Synapse, Aix-Marseille Université, 13015 Marseille, France</footnote>

<footnote><sup>b</sup> Marseille Proteomique (MaP), Plateforme Proteomique IMM, CNRS FR3479, Aix-Marseille Université, 31 Chemin Joseph Aiguier, 13009 Marseille, France</footnote>

<footnote><sup>c</sup> Department of Physiology, Cell Physiology Lab, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 03080, South Korea</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: jose-jorge.ramirez-franco@univ-amu.fr (J. Ramirez-Franco), oussama.el-far@inserm.fr (O. El Far).</footnote>
induce Kv₁.₁ downregulation underlying neuronal hyperexcitability and irregular spiking in the CA1 pyramidal cells (Zhou et al., 2023). Although LGI1 is part of the Kv₁-associated proteome (Ogawa et al., 2010) (Schulte et al., 2006) (Irani et al., 2010) (Ramirez-Franco et al., 2022), how the absence of an extracellular glycoprotein leads to the downregulation of Kv₁ subunits has not yet been elucidated. LGI1 binds to the proteolytically inactive transmembrane ADAM family members ADAM11, 22 and 23 (Sagane et al., 2008) (Yamagata et al., 2018) (Owuor et al., 2009) and Kv₁ is a constituent of the ADAM22-associated proteome (Fukata et al., 2021). While ADAM22 (Fukata et al., 2006) as well as Kv₁.₁ and Kv₁.₂-terminal tails, directly interact with Post-synaptic Density Proteins 95/93 (PSD95/93), also called Disc Large proteins 4 and 2 respectively (DLG4/2) via PDZ domains (Kim et al., 1995), the interplay between the ADAMs, PSD95/93 and Kv1 subunits is complex. PDZ interaction domains of Kv₁ are crucial for surface expression but not axonal targeting (Gu et al., 2003). Also, PSD93, but not PSD95 (Rasband et al., 2002) (Ogawa et al., 2010) nor ADAM22 (Ogawa et al., 2010), is important for Kv₁.₁ and Kv₁.₂ channel localisation and clustering at axonal initial segments (Ogawa et al., 2008).

In this study we unveil the molecular pathways by which the absence of LGI1 may trigger a decrease in the expression and mislocalization of several potassium channels at different cellular loci throughout the hippocampal formation. The use of mass spectrometry uncovered key modifications in the Kv₁ interactome that highlight the epileptogenic implication of Kv1 downregulation. We finally show that complex bursting patterns dominate in $Lgi1^{-/-}$ CA3 pyramidal neurons, and that this is associated with a substantial decrease in the somatodendritic Kv₂ channels that are known to participate in modulating spike bursting patterns (Raus Balind et al., 2019).

## 2. Materials and methods

### 2.1. Antibodies and other reagents

Mouse (K20/78; RRID:AB_10672854 and K36/15; RRID:AB_10673166) anti-Kv₁.₁, anti-Kv₁.₂ (K14/16; RRID:AB_10674277) and anti-PSD93 (N18/28; 75-057, RRID: AB_2277296) were from Antibodies Incorporated and were used for Western blots and/or immunohistofluorescence experiments. Guinea pig (386 005, RRID: AB_2737033) anti-ankyrinG (AnkG) was from Synaptic Systems and rabbit anti-Kv₂.₁ (APC-012) from Alomone labs. Monoclonal JA9 anti-PMCA4 was purchased from Thermo Fisher Scientific. Secondary Alexa coupled 488-goat anti-mouse, 488-anti-rabbit, 594-goat anti-guinea pig were from Jackson ImmunoResearch. A rabbit polyclonal anti C-terminal peptide (aa 454-468) of mouse Kv₁.₂ was produced and affinity purified by GeneCust and used for immunoprecipitation experiments, since monoclonal Kv₁.₂ antibodies gave poor immunoprecipitation yields. This antibody was used at 5 µg/ml for Western blot analysis and 5 µg / immunoprecipitated sample. HRP-coupled secondary antibodies were from Jackson ImmunoResearch. Chemicals were purchased from Euromedex and DAPI from Sigma-Aldrich.

### 2.2. Clones

pcDNA3 plasmids containing human Kv₁.₁ or rat Kv₁.₂ were a generous gift from Jeffrey Martens (University of Michigan, USA). These sequences contain pHLuorin (Kv₁.₁) and EGFP (Kv₁.₂) coding sequences inserted in frame, in the extracellular loop linking transmembrane domains one and two. EGFP sequence in Kv₁.₂ was replaced by that encoding pHLuorin, using EcoR1 and Not1 restriction sites. The full coding sequences of pHLuorine-Kv₁.₁ and pHLuorine-Kv₁.₂ were transferred into pINDUCER11 (gift from Stephen Elledge, Harvard Medical School, Boston, USA) plasmid downstream of a doxycycline inducible promotor. Kv₁.₁, Kv₁.₂, Kv₁.₃ and Kv₁.₄-terminal tail constructs were generated by standard polymerase chain reaction (PCR) using a commercial adult rat brain cDNA library (OriGene Technologies, Inc).

Sequences were cloned in pGEX-5× using EcoR I and Sal I restriction sites.

### 2.3. Bacterial recombinant protein expression and purification

GST-Kv C-terminal tail fusion constructs were transfected into the BL21 bacterial strain and recombinant protein expression induced by 0.5 mM IPTG. Bacterial pellets were resuspended and solubilised in 25 mM Tris pH 7.4, 150 mM NaCl supplemented with 1% Triton X-100 in the presence of protease inhibitors (Complete, Roche Diagnostics GmbH). Fusion proteins were purified on glutathione-Sepharose (Cytiva) using previously described procedures (Flores et al., 2019).

### 2.4. Western blots and ELISA

Western blots and ELISA experiments were performed using previously described procedures (Di Giovanni et al., 2010). For Western blots, 50 µg of proteins were loaded in each well. Signals were quantified using ImageJ and normalized to GAPDH intensities. For ELISA, 1 µg of purified GST-fusion proteins were immobilized in each well and proteins were detected using either anti-GST (1:2000) or anti-Kv₁.₂ polyclonal antibody. Anti-Kv₁.₂ signals were normalized to anti-GST signals on parallel wells. Western blots were performed on triplicate samples from at least 3 animals. Statistical analysis was performed using the two-sample *t*-test or two-tailed Mann-Whitney *U* test.

### 2.5. Cell culture

HEK293T cells were cultured at 37 °C in DMEM (GlutaMAX) supplemented with 10% foetal bovine serum, 1% non-essential amino acids and 1% streptomycin/penicillin mix. Transfection with pINDUCER11-Kv₁.₁ or pINDUCER11-Kv₁.₂ expression plasmids was performed in optiMEM using Lipofectamine 2000. Kv expression was induced by doxycycline for 48 h before cell harvesting. Cells were lysed in a Potter homogenizer using 20 mM HEPES pH 7.4, 150 mM NaCl supplemented with protease inhibitors (Complete, Roche Diagnostics GmbH). Cell membranes were harvested by a 30 min centrifugation at 16000 *xg* and resuspended in lysis buffer for protein analysis.

### 2.6. Biochemical sample preparations and immunoprecipitations

Wild type and $Lgi1^{-/-}$ (Chabrol et al., 2010) (the original $Lgi1^{-/-}$ mice strain was a generous gift from Stéphanie Baulac, ICM, Paris, France) mice total brains or hippocampi (15-16 days) were extracted, snap-frozen in liquid nitrogen and kept at -80 °C until use. Homogenisation (1 ml / brain) was performed in HB buffer (20 mM Na phosphate pH 7.4, 30 mM NaCl containing phosphatase inhibitors (Pierce™) and protease inhibitors (Complete, Roche Diagnostics GmbH) and nuclei were eliminated by 900 *xg* centrifugation. Supernatants were either directly processed for SDS-PAGE analysis and Western blot or solubilised for immunoprecipitation in HB supplemented with 1% CHAPS at 5 mg/ml and subjected to 100.000 *xg* ultracentrifugation. The solubilised material from both wildtype and $Lgi1^{-/-}$ samples was precleared with rProtein A Sepharose Fast Flow (Cytiva) and immunoprecipitated in triplicates, from three different animals, using the polyclonal anti-Kv₁.₂ antibody (3-5 µg / immunoprecipitated sample). Immunoprecipitated samples were washed three times with HB containing 0.5% CHAPS before denaturation. Samples were then analysed either by SDS-PAGE and Western blot or by mass spectrometry. C3HeB/FeJ Kcna2 mice brains (P10) ($Kv_{1.2}^{-/-}$) and their wild type littermates (Hyun et al., 2013) were extracted and deep frozen in dry ice then transferred to -80 °C and stored until use. Homogenisation and analysis by Western blot were performed as described for WT and $Lgi1^{-/-}$ samples. The number of experiments performed, as well as the associated statistics are mentioned in the legends of the corresponding figures.
induce Kv₁.₁ downregulation underlying neuronal hyperexcitability and irregular spiking in the CA1 pyramidal cells (Zhou et al., 2023). Although LGI1 is part of the Kv₁-associated proteome (Ogawa et al., 2010) (Schulte et al., 2006) (Irani et al., 2010) (Ramirez-Franco et al., 2022), how the absence of an extracellular glycoprotein leads to the downregulation of Kv₁ subunits has not yet been elucidated. LGI1 binds to the proteolytically inactive transmembrane ADAM family members ADAM11, 22 and 23 (Sagane et al., 2008) (Yamagata et al., 2018) (Owuor et al., 2009) and Kv₁ is a constituent of the ADAM22-associated proteome (Fukata et al., 2021). While ADAM22 (Fukata et al., 2006) as well as Kv₁.₁ and Kv₁.₂-terminal tails, directly interact with Post-synaptic Density Proteins 95/93 (PSD95/93), also called Disc Large proteins 4 and 2 respectively (DLG4/2) via PDZ domains (Kim et al., 1995), the interplay between the ADAMs, PSD95/93 and Kv1 subunits is complex. PDZ interaction domains of Kv₁ are crucial for surface expression but not axonal targeting (Gu et al., 2003). Also, PSD93, but not PSD95 (Rasband et al., 2002) (Ogawa et al., 2010) nor ADAM22 (Ogawa et al., 2010), is important for Kv₁.₁ and Kv₁.₂ channel localisation and clustering at axonal initial segments (Ogawa et al., 2008).

In this study we unveil the molecular pathways by which the absence of LGI1 may trigger a decrease in the expression and mislocalization of several potassium channels at different cellular loci throughout the hippocampal formation. The use of mass spectrometry uncovered key modifications in the Kv₁ interactome that highlight the epileptogenic implication of Kv1 downregulation. We finally show that complex bursting patterns dominate in $Lgi1^{-/-}$ CA3 pyramidal neurons, and that this is associated with a substantial decrease in the somatodendritic Kv₂ channels that are known to participate in modulating spike bursting patterns (Raus Balind et al., 2019).

# 2. Materials and methods

## 2.1. Antibodies and other reagents

Mouse (K20/78; RRID:AB_10672854 and K36/15; RRID:AB_10673166) anti-Kv₁.₁, anti-Kv₁.₂ (K14/16; RRID:AB_10674277) and anti-PSD93 (N18/28; 75-057, RRID: AB_2277296) were from Antibodies Incorporated and were used for Western blots and/or immunohistofluorescence experiments. Guinea pig (386 005, RRID: AB_2737033) anti-ankyrinG (AnkG) was from Synaptic Systems and rabbit anti-Kv₂.₁ (APC-012) from Alomone labs. Monoclonal JA9 anti-PMCA4 was purchased from Thermo Fisher Scientific. Secondary Alexa coupled 488-goat anti-mouse, 488-anti-rabbit, 594-goat anti-guinea pig were from Jackson ImmunoResearch. A rabbit polyclonal anti C-terminal peptide (aa 454-468) of mouse Kv₁.₂ was produced and affinity purified by GeneCust and used for immunoprecipitation experiments, since monoclonal Kv₁.₂ antibodies gave poor immunoprecipitation yields. This antibody was used at 5 µg/ml for Western blot analysis and 5 µg / immunoprecipitated sample. HRP-coupled secondary antibodies were from Jackson ImmunoResearch. Chemicals were purchased from Euromedex and DAPI from Sigma-Aldrich.

## 2.2. Clones

pcDNA3 plasmids containing human Kv₁.₁ or rat Kv₁.₂ were a generous gift from Jeffrey Martens (University of Michigan, USA). These sequences contain pHLuorin (Kv₁.₁) and EGFP (Kv₁.₂) coding sequences inserted in frame, in the extracellular loop linking transmembrane domains one and two. EGFP sequence in Kv₁.₂ was replaced by that encoding pHLuorin, using EcoR1 and Not1 restriction sites. The full coding sequences of pHLuorine-Kv₁.₁ and pHLuorine-Kv₁.₂ were transferred into pINDUCER11 (gift from Stephen Elledge, Harvard Medical School, Boston, USA) plasmid downstream of a doxycycline inducible promotor. Kv₁.₁, Kv₁.₂, Kv₁.₃ and Kv₁.₄-terminal tail constructs were generated by standard polymerase chain reaction (PCR) using a commercial adult rat brain cDNA library (OriGene Technologies, Inc).

Sequences were cloned in pGEX-5× using EcoR I and Sal I restriction sites.

## 2.3. Bacterial recombinant protein expression and purification

GST-Kv C-terminal tail fusion constructs were transfected into the BL21 bacterial strain and recombinant protein expression induced by 0.5 mM IPTG. Bacterial pellets were resuspended and solubilised in 25 mM Tris pH 7.4, 150 mM NaCl supplemented with 1% Triton X-100 in the presence of protease inhibitors (Complete, Roche Diagnostics GmbH). Fusion proteins were purified on glutathione-Sepharose (Cytiva) using previously described procedures (Flores et al., 2019).

## 2.4. Western blots and ELISA

Western blots and ELISA experiments were performed using previously described procedures (Di Giovanni et al., 2010). For Western blots, 50 µg of proteins were loaded in each well. Signals were quantified using ImageJ and normalized to GAPDH intensities. For ELISA, 1 µg of purified GST-fusion proteins were immobilized in each well and proteins were detected using either anti-GST (1:2000) or anti-Kv₁.₂ polyclonal antibody. Anti-Kv₁.₂ signals were normalized to anti-GST signals on parallel wells. Western blots were performed on triplicate samples from at least 3 animals. Statistical analysis was performed using the two-sample *t*-test or two-tailed Mann-Whitney *U* test.

## 2.5. Cell culture

HEK293T cells were cultured at 37 °C in DMEM (GlutaMAX) supplemented with 10% foetal bovine serum, 1% non-essential amino acids and 1% streptomycin/penicillin mix. Transfection with pINDUCER11-Kv₁.₁ or pINDUCER11-Kv₁.₂ expression plasmids was performed in optiMEM using Lipofectamine 2000. Kv expression was induced by doxycycline for 48 h before cell harvesting. Cells were lysed in a Potter homogenizer using 20 mM HEPES pH 7.4, 150 mM NaCl supplemented with protease inhibitors (Complete, Roche Diagnostics GmbH). Cell membranes were harvested by a 30 min centrifugation at 16000 *xg* and resuspended in lysis buffer for protein analysis.

## 2.6. Biochemical sample preparations and immunoprecipitations

Wild type and $Lgi1^{-/-}$ (Chabrol et al., 2010) (the original $Lgi1^{-/-}$ mice strain was a generous gift from Stéphanie Baulac, ICM, Paris, France) mice total brains or hippocampi (15-16 days) were extracted, snap-frozen in liquid nitrogen and kept at -80 °C until use. Homogenisation (1 ml / brain) was performed in HB buffer (20 mM Na phosphate pH 7.4, 30 mM NaCl containing phosphatase inhibitors (Pierce™) and protease inhibitors (Complete, Roche Diagnostics GmbH) and nuclei were eliminated by 900 *xg* centrifugation. Supernatants were either directly processed for SDS-PAGE analysis and Western blot or solubilised for immunoprecipitation in HB supplemented with 1% CHAPS at 5 mg/ml and subjected to 100.000 *xg* ultracentrifugation. The solubilised material from both wildtype and $Lgi1^{-/-}$ samples was precleared with rProtein A Sepharose Fast Flow (Cytiva) and immunoprecipitated in triplicates, from three different animals, using the polyclonal anti-Kv₁.₂ antibody (3-5 µg / immunoprecipitated sample). Immunoprecipitated samples were washed three times with HB containing 0.5% CHAPS before denaturation. Samples were then analysed either by SDS-PAGE and Western blot or by mass spectrometry. C3HeB/FeJ Kcna2 mice brains (P10) ($Kv_{1.2}^{-/-}$) and their wild type littermates (Hyun et al., 2013) were extracted and deep frozen in dry ice then transferred to -80 °C and stored until use. Homogenisation and analysis by Western blot were performed as described for WT and $Lgi1^{-/-}$ samples. The number of experiments performed, as well as the associated statistics are mentioned in the legends of the corresponding figures.
## 2.7. Mass spectrometry

Samples were treated and analysed as in (Ramirez-Franco et al., 2022). Briefly, immunoprecipitated proteins were concentrated and analysed by mass spectrometry using a hybrid Q-Orbitrap mass spectrometer (Q-Exactive, Thermo Fisher Scientific, United States) coupled to nanoliquid chromatography (LC) Dionex RSLC Ultimate 3000 system (Thermo Fisher Scientific, United States). All raw data files generated by MS were processed to generate mgf files and screened against the Swissprot database (Swissprot version June 2019, (560,118 sequence), with the taxonomy *Mus musculus*, using the MASCOT software (www matrixscience.com, version 2.3). The database was searched in the decoy mode and emPAIs automatically calculated by the MASCOT algorithm. All samples were analysed in triplicates. For each antibody, background interactomes obtained with non-immune antibodies were subtracted using an Excel script. Keratins, ribosomal proteins, DNA binding proteins, mitochondrial proteins, transcriptional proteins and immunoglobulins were excluded from the list of candidates. Candidates with emPAI <0.1 were not interpreted. Functional properties of candidate proteins were classified using the pantherdb web site (www.pant herdb.org) (Supplementary sheet). The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2022) partner repository with the dataset identifier PXD043649.

## 2.8. Immunohistofluorescence staining of fixed brains

All experiments were performed in accordance with the European and institutional guidelines for the care and use of laboratory animals (Council Directive 86/609/EEC and French National Research Council) and approved by the local authority (Préfecture des Bouches-du-Rhône, Marseille). Mice of either sex were used in this study. Fixed brains of P14-P16 C57BL/6 wild-type mice or *Lgi1*⁻/⁻ littermates of either sex were sliced and stained as described in (Ramirez-Franco et al., 2022). For Kv1.1 and Kv1.2 staining, an antigen retrieval process was applied as in (Haddjeri-Hopkins et al., 2021) (Jiao et al., 1999). Three animals per genotype were used for each of the immunohistofluorescence conditions tested.

## 2.9. Image analysis

Image analysis was performed as described in (Ramirez-Franco et al., 2022). Briefly, AnkyrinG staining was used to identify the AIS and hence to measure the immunoreactivity of Kv1.1, Kv1.2, Kv2.1 and PSD93 in this compartment. For 3D quantification in confocal images, 3D objects counter (Bolte and Cordelieres, 2006) and 3D ROI manager (Ollion et al., 2013) were integrated into custom-written FJI scripts available upon reasonable request. For the sake of clarity in Fig. 4E, the PSD93 signal corresponding to the AIS was extracted using the “Clear Outside” method in ImageJ after using the thresholded AnkyrinG signal as a 3D selection.

## 2.10. Acute slice preparation

Postnatal (P14) WT or *Lgi1*⁻/⁻ mice were deeply anesthetized in isoflurane (3% in oxygen, 2l/min) and decapitated. After dissection, the brains were immersed in oxygenated (95% O₂, 5% CO₂) ice-cold low-calcium artificial CSF (aCSF) containing the following (in mM): 125 NaCl; 2.5 KCl; 1.25 NaH₂PO₄; 0.5 CaCl₂; 2 MgCl₂; 26 NaHCO₃; 10 d-Glucose; pH 7.4. Horizontal slices (350 µm) were cut in a vibratome (Leica VT1200S) in oxygenated ice-cold low-calcium aCSF. Afterwards, slices were transferred and kept at least 1 h at RT in oxygenated aCSF containing the following (in mM): 125 NaCl; 2.5 KCl; 0.8 NaH₂PO₄; 26 NaHCO₃; 3 CaCl₂; 2 MgCl₂; 10 d-Glucose; pH 7.4.

## 2.11. Electrophysiology and data analysis

Whole-cell recordings of visually identified CA3 pyramidal neurons were performed in a temperature controlled (30 °C) chamber superfused with oxygenated aCSF. Borosilicate patch pipettes (3–5 MΩ) were pulled on a DMZ-Universal Puller (Zeitz Instruments) and filled with an internal solution containing (in mM): 120 K-gluconate; 20 KCl; 10 HEPES; 0.1 EGTA; 2 MgCl₂; 2 Na₂ATP; 0.3 NaGTP; pH 7.4; 290–300 mOsm. Recordings were made in current clamp mode with a multiclamp 700B amplifier (Molecular Devices) and pClamp10 software. Recordings were acquired at 10 kHz and filtered at 3 kHz. Neurons with a resting membrane potential > -55 mV were discarded from the final analysis. Intrinsic excitability was assessed by injecting a range of 0.8 s current pulses (ranging from -50 to 250 pA; 10 pA / step) and counting the number of spikes. Input-output curves were determined and the rheobase was defined as the minimal current eliciting at least one action potential. To study bursting propensity, depolarizing current injections of 0.5 s (300 or 600 pA) were applied every 2 s. Bursts were scored when ≥2 APs appeared in the same response showing progressively smaller amplitude and longer duration (Raus Balind et al., 2019). To analyse the proportion of cells exhibiting complex bursts, only cells showing bursts at the rheobase current injection were considered. To study burst propensity, only cells exhibiting bursts when challenged with either 300 pA or 600 pA depolarizing pulses were defined as positive. AP parameters (AP half-width and AP amplitude) were extracted from the first spike in the rheobase. To prevent synaptic contamination 3 mM kynurenate and 100 µM picrotoxin (both from SIGMA) were systematically added to the aCSF. Cells were held at ~ -70 mV during experiments and recordings were not corrected for liquid junction potential. All data were analysed using ClampFit (Molecular Devices). Data are presented as mean ± SEM. Mann-Whitney U test, Two samples t-test, and Fisher's exact test were used for comparisons. Normality tests were applied before the use of the Mann-Whitney U test or Two samples t-test. The number of experiments performed, as well as the associated statistics are mentioned in the legends of the corresponding figures. For rheobase analyses WT *n* = 30 cells from 8 animals; KO *n* = 35 cells from 8 animals. For bursting propensity analyses WT *n* = 8 cells from 2 animals; KO *n* = 8 cells from 4 animals.

# 3. Results

## 3.1. *Kv1.1* and *Kv1.2* expression in *Lgi1*⁻/⁻

We have previously shown that Kv1.1 and Kv1.2 subunits were massively downregulated in LGI1 knock-out mice (*Lgi1*⁻/⁻) total brain extracts and that, in this genetic background, Kv1.1 was almost absent from axonal initial segments of hippocampal and cortical neurons (Seagar et al., 2017). In the light of the major enrichment of LGI1 in the hippocampal formation (Ramirez-Franco et al., 2022), we compared by Western blot the downregulation level of Kv1.1 and Kv1.2 subunits in total brain homogenates as well as in hippocampal extracts (Figs. 1 and 2). The decrease in expression of Kv1.1 and Kv1.2 in total brain (Fig. 1A) (Kv1.1 WT 1.00 ± 0.07 vs Kv1.1 *Lgi1*⁻/⁻ 0.72 ± 0.08, *p* = 0.047; Kv1.2 WT 1.00 ± 0.05 vs Kv1.2 *Lgi1*⁻/⁻ 0.52 ± 0.04, *p* = 1.03 × 10⁻⁷) was less severe than in hippocampus (Fig. 1B, Kv1.1 WT 1.00 ± 0.05 vs Kv1.1 *Lgi1*⁻/⁻ 0.55 ± 0.03, *p* = 1.33 × 10⁻⁷; Kv1.2 WT 1.00 ± 0.04 vs Kv1.2 *Lgi1*⁻/⁻ 0.38 ± 0.02, *p* = 1.73 × 10⁻¹³).

In order to address Kv₁ expression and distribution profile, we investigated, the detailed comparative immunofluorescence staining of Kv1.1 and Kv1.2 in the hippocampus in both WT and *Lgi1*⁻/⁻ backgrounds. It is noteworthy that conventional immunohistochemical techniques could render the epitopes of Kv1.1 and Kv1.2 inaccessible to antibodies, especially in slices from prefixed brain, precluding detection of these channels particularly in the AIS of hippocampal neurons (Lorincz and Nusser, 2008b). In our hands, this proved to be the case, since Kv1.1 was not detected in the absence of antigen retrieval protocols
## 2.7. Mass spectrometry

Samples were treated and analysed as in (Ramirez-Franco et al., 2022). Briefly, immunoprecipitated proteins were concentrated and analysed by mass spectrometry using a hybrid Q-Orbitrap mass spectrometer (Q-Exactive, Thermo Fisher Scientific, United States) coupled to nanoliquid chromatography (LC) Dionex RSLC Ultimate 3000 system (Thermo Fisher Scientific, United States). All raw data files generated by MS were processed to generate mgf files and screened against the Swissprot database (Swissprot version June 2019, (560,118 sequence), with the taxonomy *Mus musculus*, using the MASCOT software (www matrixscience.com, version 2.3). The database was searched in the decoy mode and emPAIs automatically calculated by the MASCOT algorithm. All samples were analysed in triplicates. For each antibody, background interactomes obtained with non-immune antibodies were subtracted using an Excel script. Keratins, ribosomal proteins, DNA binding proteins, mitochondrial proteins, transcriptional proteins and immunoglobulins were excluded from the list of candidates. Candidates with emPAI <0.1 were not interpreted. Functional properties of candidate proteins were classified using the pantherdb web site (www.pant herdb.org) (Supplementary sheet). The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2022) partner repository with the dataset identifier PXD043649.

## 2.8. Immunohistofluorescence staining of fixed brains

All experiments were performed in accordance with the European and institutional guidelines for the care and use of laboratory animals (Council Directive 86/609/EEC and French National Research Council) and approved by the local authority (Préfecture des Bouches-du-Rhône, Marseille). Mice of either sex were used in this study. Fixed brains of P14-P16 C57BL/6 wild-type mice or *Lgi1*<sup>-/-</sup> littermates of either sex were sliced and stained as described in (Ramirez-Franco et al., 2022). For Kv1.1 and Kv1.2 staining, an antigen retrieval process was applied as in (Haddjeri-Hopkins et al., 2021) (Jiao et al., 1999). Three animals per genotype were used for each of the immunohistofluorescence conditions tested.

## 2.9. Image analysis

Image analysis was performed as described in (Ramirez-Franco et al., 2022). Briefly, AnkyrinG staining was used to identify the AIS and hence to measure the immunoreactivity of Kv1.1, Kv1.2, Kv2.1 and PSD93 in this compartment. For 3D quantification in confocal images, 3D objects counter (Bolte and Cordelieres, 2006) and 3D ROI manager (Ollion et al., 2013) were integrated into custom-written FJI scripts available upon reasonable request. For the sake of clarity in Fig. 4E, the PSD93 signal corresponding to the AIS was extracted using the “Clear Outside” method in ImageJ after using the thresholded AnkyrinG signal as a 3D selection.

## 2.10. Acute slice preparation

Postnatal (P14) WT or *Lgi1*<sup>-/-</sup> mice were deeply anesthetized in isoflurane (3% in oxygen, 2l/min) and decapitated. After dissection, the brains were immersed in oxygenated (95% O₂, 5% CO₂) ice-cold low-calcium artificial CSF (aCSF) containing the following (in mM): 125 NaCl; 2.5 KCl; 1.25 NaH₂PO₄; 0.5 CaCl₂; 2 MgCl₂; 26 NaHCO₃; 10 d-Glucose; pH 7.4. Horizontal slices (350 µm) were cut in a vibratome (Leica VT1200S) in oxygenated ice-cold low-calcium aCSF. Afterwards, slices were transferred and kept at least 1 h at RT in oxygenated aCSF containing the following (in mM): 125 NaCl; 2.5 KCl; 0.8 NaH₂PO₄; 26 NaHCO₃; 3 CaCl₂; 2 MgCl₂; 10 d-Glucose; pH 7.4.

## 2.11. Electrophysiology and data analysis

Whole-cell recordings of visually identified CA3 pyramidal neurons were performed in a temperature controlled (30 °C) chamber superfused with oxygenated aCSF. Borosilicate patch pipettes (3–5 MΩ) were pulled on a DMZ-Universal Puller (Zeitz Instruments) and filled with an internal solution containing (in mM): 120 K-gluconate; 20 KCl; 10 HEPES; 0.1 EGTA; 2 MgCl₂; 2 Na₂ATP; 0.3 NaGTP; pH 7.4; 290–300 mOsm. Recordings were made in current clamp mode with a multiclamp 700B amplifier (Molecular Devices) and pClamp10 software. Recordings were acquired at 10 kHz and filtered at 3 kHz. Neurons with a resting membrane potential > -55 mV were discarded from the final analysis. Intrinsic excitability was assessed by injecting a range of 0.8 s current pulses (ranging from -50 to 250 pA; 10 pA / step) and counting the number of spikes. Input-output curves were determined and the rheobase was defined as the minimal current eliciting at least one action potential. To study bursting propensity, depolarizing current injections of 0.5 s (300 or 600 pA) were applied every 2 s. Bursts were scored when ≥2 APs appeared in the same response showing progressively smaller amplitude and longer duration (Raus Balind et al., 2019). To analyse the proportion of cells exhibiting complex bursts, only cells showing bursts at the rheobase current injection were considered. To study burst propensity, only cells exhibiting bursts when challenged with either 300 pA or 600 pA depolarizing pulses were defined as positive. AP parameters (AP half-width and AP amplitude) were extracted from the first spike in the rheobase. To prevent synaptic contamination 3 mM kynurenate and 100 µM picrotoxin (both from SIGMA) were systematically added to the aCSF. Cells were held at ~ -70 mV during experiments and recordings were not corrected for liquid junction potential. All data were analysed using ClampFit (Molecular Devices). Data are presented as mean ± SEM. Mann-Whitney U test, Two samples t-test, and Fisher's exact test were used for comparisons. Normality tests were applied before the use of the Mann-Whitney U test or Two samples t-test. The number of experiments performed, as well as the associated statistics are mentioned in the legends of the corresponding figures. For rheobase analyses WT *n* = 30 cells from 8 animals; KO *n* = 35 cells from 8 animals. For bursting propensity analyses WT *n* = 8 cells from 2 animals; KO *n* = 8 cells from 4 animals.

# 3. Results

## 3.1. *Kv1.1* and *Kv1.2* expression in *Lgi1*<sup>-/-</sup>

We have previously shown that Kv1.1 and Kv1.2 subunits were massively downregulated in LGI1 knock-out mice (*Lgi1*<sup>-/-</sup>) total brain extracts and that, in this genetic background, Kv1.1 was almost absent from axonal initial segments of hippocampal and cortical neurons (Seagar et al., 2017). In the light of the major enrichment of LGI1 in the hippocampal formation (Ramirez-Franco et al., 2022), we compared by Western blot the downregulation level of Kv1.1 and Kv1.2 subunits in total brain homogenates as well as in hippocampal extracts (Figs. 1 and 2). The decrease in expression of Kv1.1 and Kv1.2 in total brain (Fig. 1A) (Kv1.1 WT 1.00 ± 0.07 vs Kv1.1 *Lgi1*<sup>-/-</sup> 0.72 ± 0.08, *p* = 0.047; Kv1.2 WT 1.00 ± 0.05 vs Kv1.2 *Lgi1*<sup>-/-</sup> 0.52 ± 0.04, *p* = 1.03 × 10<sup>-7</sup>) was less severe than in hippocampus (Fig. 1B, Kv1.1 WT 1.00 ± 0.05 vs Kv1.1 *Lgi1*<sup>-/-</sup> 0.55 ± 0.03, *p* = 1.33 × 10<sup>-7</sup>; Kv1.2 WT 1.00 ± 0.04 vs Kv1.2 *Lgi1*<sup>-/-</sup> 0.38 ± 0.02, *p* = 1.73 × 10<sup>-13</sup>).

In order to address Kv₁ expression and distribution profile, we investigated, the detailed comparative immunofluorescence staining of Kv₁.1 and Kv₁.2 in the hippocampus in both WT and *Lgi1*<sup>-/-</sup> backgrounds. It is noteworthy that conventional immunohistochemical techniques could render the epitopes of Kv1.1 and Kv1.2 inaccessible to antibodies, especially in slices from prefixed brain, precluding detection of these channels particularly in the AIS of hippocampal neurons (Lorincz and Nusser, 2008b). In our hands, this proved to be the case, since Kv1.1 was not detected in the absence of antigen retrieval protocols
<figure><img src="image_4.png" /><figcaption>Fig. 1. Kv₁.₁ expression is reduced in Lgi1⁻/⁻ mice.</figcaption></figure>

(A) Quantification by Western blot of Kv₁.₁ expression in whole brain homogenates of WT (1.00 ± 0.07) and Lgi1⁻/⁻ (0.72 ± 0.08), p = 0.047; n = 4; Two-sample t-test. (B) Quantification by Western blot of Kv₁.₁ expression in hippocampal homogenates of WT (1.00 ± 0.05) and Lgi1⁻/⁻ (0.55 ± 0.03); p = 1.33 × 10⁻⁷; n = 16; Two-sample t-test. In a and b, signals were normalized to GAPDH expression levels. (C) Representative AISs of CA3 pyramidal cells (see Supplementary Fig. S1 for larger fields of view) highlighting the necessity of antigen retrieval (bottom panels) for Kv₁.₁ detection in the hippocampus. Scale bar 5 µm (D) Representative images of Kv₁.₁ immunoreactivity (IR) across CA1 (top panels, Scale bar = 50 µm), CA3 (middle panels, Scale bar = 50 µm), and DG (bottom panels, Scale bar = 50 µm) in WT vs Lgi1⁻/⁻ mouse. SO: Str. Oriens; SP: Str. Pyramidale; SR: Str. Radiatum; SL: Str. Lucidum; ML: Mol. Layer; GCL: Granule Cell Layer; Hi: Hilus. (E) Quantification of the differences in the normalized Kv₁.₁ IR (to WT levels) through the different hippocampal strata. For the sake of clarity, only non-significant comparisons are indicated. For all other comparison \*\*p < 0.01 (CA1-SO WT = 1.00 ± 0.02 vs Lgi1⁻/⁻ = 0.66 ± 0.02 p = 5.96 × 10⁻¹³; CA1-SR WT = 1.00 ± 0.03 vs Lgi1⁻/⁻ = 0.05 ± 0.05 p = 1.20 × 10⁻⁶; CA3-SO WT = 1.00 ± 0.04 vs Lgi1⁻/⁻ = 0.53 ± 0.03 p = 2.75 × 10⁻¹⁰; CA3-SL WT = 1.00 ± 0.05 vs Lgi1⁻/⁻ = 0.88 ± 0.05 p = 0.07; CA3-SR WT = 1.00 ± 0.03 vs Lgi1⁻/⁻ = 0.39 ± 0.02 p = 2.34 × 10⁻¹⁰; DG-ML-SPB WT = 1.00 ± 0.02 vs Lgi1⁻/⁻ = 0.70 ± 0.03 p = 1.20 × 10⁻⁸; DG-Hilus WT = 1.00 ± 0.04 vs Lgi1⁻/⁻ = 0.65 ± 0.03 p = 3.57 × 10⁻⁸; DG-ML-IPB WT = 1.00 ± 0.06 vs Lgi1⁻/⁻ = 0.58 ± 0.03 p = 1.03 × 10⁻⁷; Two-sample t-test). (F) Left, 63× images of Kv₁.₁ immunoreactivity at CA3 AIS in WT (top panels) and Lgi1⁻/⁻ (bottom panels). Scale bar = 10 µm. Right, detail of a CA3 pyramidal neuron AIS in WT (top panels) and Lgi1⁻/⁻ (bottom panels). Scale bar = 2.5 µm. (G) 63× images showing no differences in Kv₁.₁ IR at the AIS of PV⁺ interneurons delimited by yellow arrowheads. Of note, the Kv₁ signal in the surrounding pyramidal neurons AIS (dotted-line rectangles) is severely decreased. Scale bar = 10 µm (H) Cumulative probability plots of Kv₁.₁ immunoreactivity at individual AIS of WT (black squares) and Lgi1⁻/⁻ (red circles) CA3 pyramidal neurons (WT Pyr CA3 neurons = 1.00 ± 0.02 vs Lgi1⁻/⁻ CA3 Pyr neurons = 0.23 ± 0.01; p = 0.00, Kolmogorov-Smirnov Test). (I) Comparisons of Kv₁.₁ immunoreactivity (Normalized to WT CA3 Pyr neurons) at the AIS of pyramidal cells (dark tones) and PV⁺ interneurons (light tones) across WT (black) and Lgi1⁻/⁻ (red) hippocampi (Pyr WT = 1.00 ± 0.02 vs Pyr Lgi1⁻/⁻ = 0.40 ± 0.01 p = 2.43 × 10⁻¹³²; PV⁺ IN WT = 1.25 ± 0.10 vs PV⁺ IN Lgi1⁻/⁻ = 1.22 ± 0.08; p = 1.00; ANOVA followed by Bonferroni's test for means comparison).

(Fig. 1C and Supplementary Fig. S1). The use of the same antibody on organotypic slices (Extremet et al., 2023) or using mild fixation conditions in thin slices (Petit-Pedrol et al., 2018) could facilitate epitope access. Due to the tortuosity of the AIS in CA3, we chose to use 50 µm thick sections and therefore employed antigen retrieval techniques (Jiao et al., 1999) (Haddjeri-Hopkins et al., 2021) to address Kv₁.₁ (Fig. 1C-H) and Kv₁.₂ (Fig. 2C-H) distribution. In addition to the previously reported decrease in Kv₁ expression in the AIS (Fig. 1E and Fig. 2E), we observed an overall decrease in all hippocampal strata (CA1, CA3 and DG) (Fig. 1C, D, G and Fig. 2C, D, G) with a more prominent decrease of Kv₁.₂ versus Kv₁.₁. Interestingly, we observed that in parvalbumin (PV) positive interneurons, the expression levels of both Kv₁.₁ and Kv₁.₂ subunits were not affected compared to the surrounding pyramidal neurons AIS,

(Fig. 1F, H and Fig. 2F, H) suggesting that Kv₁ expression levels in PV⁺ cells are not regulated by LGI1.

## 3.2. Characterization of Kv₁.₂ antibodies used for immunoprecipitation

In order to address changes in the Kv₁.₂ proteome between WT and Lgi1⁻/⁻, we generated a polyclonal antibody directed against a C-terminal peptide sequence and performed immunoprecipitations followed by mass spectrometry analysis. As shown in Fig. 3B, this antibody recognizes Kv₁.₂ from brain extracts as well as recombinant pHLuorin-tagged full length Kv₁.₂, but not Kv₁.₁ expressed in HEK cells. However, the band recognized in wild-type mice brain extracts was still present in Kv₁.₂ knock out extracts (Fig. 3C) suggesting that this antibody can
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Kv₁.₁ expression is reduced in Lgi1⁻/⁻ mice.</figcaption></figure>

(A) Quantification by Western blot of Kv₁.₁ expression in whole brain homogenates of WT (1.00 ± 0.07) and Lgi1⁻/⁻ (0.72 ± 0.08), p = 0.047; n = 4; Two-sample t-test. (B) Quantification by Western blot of Kv₁.₁ expression in hippocampal homogenates of WT (1.00 ± 0.05) and Lgi1⁻/⁻ (0.55 ± 0.03); p = 1.33 × 10⁻⁷; n = 16; Two-sample t-test. In a and b, signals were normalized to GAPDH expression levels. (C) Representative AISs of CA3 pyramidal cells (see Supplementary Fig. S1 for larger fields of view) highlighting the necessity of antigen retrieval (bottom panels) for Kv₁.₁ detection in the hippocampus. Scale bar 5 µm (D) Representative images of Kv₁.₁ immunoreactivity (IR) across CA1 (top panels, Scale bar = 50 µm), CA3 (middle panels, Scale bar = 50 µm), and DG (bottom panels, Scale bar = 50 µm) in WT vs Lgi1⁻/⁻ mouse. SO: Str. Oriens; SP: Str. Pyramidale; SR: Str. Radiatum; SL: Str. Lucidum; ML: Mol. Layer; GCL: Granule Cell Layer; Hi: Hilus. (E) Quantification of the differences in the normalized Kv₁.₁ IR (to WT levels) through the different hippocampal strata. For the sake of clarity, only non-significant comparisons are indicated. For all other comparison \*\*p < 0.01 (CA1-SO WT = 1.00 ± 0.02 vs Lgi1⁻/⁻ = 0.66 ± 0.02 p = 5.96 × 10⁻¹³; CA1-SR WT = 1.00 ± 0.03 vs Lgi1⁻/⁻ = 0.05 ± 0.05 p = 1.20 × 10⁻⁶; CA3-SO WT = 1.00 ± 0.04 vs Lgi1⁻/⁻ = 0.53 ± 0.03 p = 2.75 × 10⁻¹⁰; CA3-SL WT = 1.00 ± 0.05 vs Lgi1⁻/⁻ = 0.88 ± 0.05 p = 0.07; CA3-SR WT = 1.00 ± 0.03 vs Lgi1⁻/⁻ = 0.39 ± 0.02 p = 2.34 × 10⁻¹⁰; DG-ML-SPB WT = 1.00 ± 0.02 vs Lgi1⁻/⁻ = 0.70 ± 0.03 p = 1.20 × 10⁻⁸; DG-Hilus WT = 1.00 ± 0.04 vs Lgi1⁻/⁻ = 0.65 ± 0.03 p = 3.57 × 10⁻⁸; DG-ML-IPB WT = 1.00 ± 0.06 vs Lgi1⁻/⁻ = 0.58 ± 0.03 p = 1.03 × 10⁻⁷; Two-sample t-test). (F) Left, 63× images of Kv₁.₁ immunoreactivity at CA3 AIS in WT (top panels) and Lgi1⁻/⁻ (bottom panels). Scale bar = 10 µm. Right, detail of a CA3 pyramidal neuron AIS in WT (top panels) and Lgi1⁻/⁻ (bottom panels). Scale bar = 2.5 µm. (G) 63× images showing no differences in Kv₁.₁ IR at the AIS of PV⁺ interneurons delimited by yellow arrowheads. Of note, the Kv₁ signal in the surrounding pyramidal neurons AIS (dotted-line rectangles) is severely decreased. Scale bar = 10 µm (H) Cumulative probability plots of Kv₁.₁ immunoreactivity at individual AIS of WT (black squares) and Lgi1⁻/⁻ (red circles) CA3 pyramidal neurons (WT Pyr CA3 neurons = 1.00 ± 0.02 vs Lgi1⁻/⁻ CA3 Pyr neurons = 0.23 ± 0.01; p = 0.00, Kolmogorov-Smirnov Test). (I) Comparisons of Kv₁.₁ immunoreactivity (Normalized to WT CA3 Pyr neurons) at the AIS of pyramidal cells (dark tones) and PV⁺ interneurons (light tones) across WT (black) and Lgi1⁻/⁻ (red) hippocampi (Pyr WT = 1.00 ± 0.02 vs Pyr Lgi1⁻/⁻ = 0.40 ± 0.01 p = 2.43 × 10⁻¹³²; PV⁺ IN WT = 1.25 ± 0.10 vs PV⁺ IN Lgi1⁻/⁻ = 1.22 ± 0.08; p = 1.00; ANOVA followed by Bonferroni's test for means comparison).

(Fig. 1C and Supplementary Fig. S1). The use of the same antibody on organotypic slices (Extremet et al., 2023) or using mild fixation conditions in thin slices (Petit-Pedrol et al., 2018) could facilitate epitope access. Due to the tortuosity of the AIS in CA3, we chose to use 50 µm thick sections and therefore employed antigen retrieval techniques (Jiao et al., 1999) (Haddjeri-Hopkins et al., 2021) to address Kv₁.₁ (Fig. 1C-H) and Kv₁.₂ (Fig. 2C-H) distribution. In addition to the previously reported decrease in Kv₁ expression in the AIS (Fig. 1E and Fig. 2E), we observed an overall decrease in all hippocampal strata (CA1, CA3 and DG) (Fig. 1C, D, G and Fig. 2C, D, G) with a more prominent decrease of Kv₁.₂ versus Kv₁.₁. Interestingly, we observed that in parvalbumin (PV) positive interneurons, the expression levels of both Kv₁.₁ and Kv₁.₂ subunits were not affected compared to the surrounding pyramidal neurons AIS,

(Fig. 1F, H and Fig. 2F, H) suggesting that Kv₁ expression levels in PV⁺ cells are not regulated by LGI1.

## 3.2. Characterization of Kv₁.₂ antibodies used for immunoprecipitation

In order to address changes in the Kv₁.₂ proteome between WT and Lgi1⁻/⁻, we generated a polyclonal antibody directed against a C-terminal peptide sequence and performed immunoprecipitations followed by mass spectrometry analysis. As shown in Fig. 3B, this antibody recognizes Kv₁.₂ from brain extracts as well as recombinant pHLuorin-tagged full length Kv₁.₂, but not Kv₁.₁ expressed in HEK cells. However, the band recognized in wild-type mice brain extracts was still present in Kv₁.₂ knock out extracts (Fig. 3C) suggesting that this antibody can
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Kv₁.₂ expression is reduced in the *Lgi1*⁻/⁻ mice.</figcaption></figure>

(A) Quantification by Western blot of Kv₁.₂ expression in brain homogenates of WT (1.00 ± 0.06) and Lgi1⁻/⁻ (0.52 ± 0.04), *p* = 1.03 × 10⁻⁷; *n* = 17; Two-sample t-test. (B) Quantification by Western blot of Kv₁.₂ in in hippocampal homogenates of WT (1.00 ± 0.04) and Lgi1⁻/⁻ (0.38 ± 0.02); *p* = 1.73 × 10⁻¹³; *n* = 20; Two-sample t-test. In a and b, signals were normalized to GAPDH expression levels. (C) representative images of Kv₁.₂ immunoreactivity (IR) across CA1 (top panels, Scale bar = 50 µm), CA3 (middle panels, Scale bar = 50 µm), and DG (bottom panels, Scale bar = 50 µm) in WT vs Lgi1⁻/⁻ mouse. SO: Str. Oriens; SP: Str. Pyramidale; SR: Str. Radiatum; SL: Str. Lucidum; ML: Mol. Layer; GCL: Granule Cell Layer; Hi: Hilus. (D) Quantification of the differences in the normalized Kv₁.₂ IR (to WT levels) through the different hippocampal strata, all comparisons are ***p* < 0.01 (CA1-SO WT = 1.00 ± 0.04 vs Lgi1⁻/⁻ = 0.52 ± 0.02 *p* = 1.58 × 10⁻¹³; CA1-SR WT = 1.00 ± 0.06 vs Lgi1⁻/⁻ = 0.46 ± 0.02 *p* = 8.37 × 10⁻¹⁰; CA3-SO WT = 1.00 ± 0.06 vs Lgi1⁻/⁻ = 0.38 ± 0.02 *p* = 6.32 × 10⁻¹¹; CA3-SL WT = 1.00 ± 0.04 vs Lgi1⁻/⁻ = 0.53 ± 0.03 *p* = 9.61 × 10⁻¹³; CA3-SR WT = 1.00 ± 0.06 vs Lgi1⁻/⁻ = 0.34 ± 0.02 *p* = 7.49 × 10⁻¹¹; DG-ML-SPB WT = 1.00 ± 0.05 vs Lgi1⁻/⁻ = 0.49 ± 0.02 *p* = 1.92 × 10⁻¹¹; DG-Hilus WT = 1.00 ± 0.05 vs Lgi1⁻/⁻ = 0.37 ± 0.01 *p* = 4.15 × 10⁻¹²; DG-ML-IPB WT = 1.00 ± 0.05 vs Lgi1⁻/⁻ = 0.46 ± 0.02 *p* = 1.83 × 10⁻¹⁰; Two-sample t-test. (E) Left, 63× images of Kv1.2 immunoreactivity at CA3 Axonal Initial segments in WT (top panels) and Lgi1⁻/⁻ (bottom panels). Scale bar = 10 µm. Right, detail of a CA3 pyramidal neuron AIS in WT (top panels) and Lgi1⁻/⁻ (bottom panels). Scale bar = 2.5 µm. (F) 63× images showing no differences in Kv₁.₂ IR at the AIS of PV⁺ interneurons. Scale bar = 10 µm (G) Cumulative probability plots of Kv₁.₂ immunoreactivity at individual AIS of WT (black squares) and Lgi1⁻/⁻ (red circles) CA3 pyramidal neurons (WT Pyr CA3 neurons = 1.00 ± 0.01 vs Lgi1⁻/⁻ CA3 Pyr neurons = 0.41 ± 0.01; *p* = 0.00, Kolmogorov-Smirnov Test). (H) Comparisons of Kv₁.₂ immunoreactivity (Normalized to WT CA3 Pyr Neurons) at the AIS of pyramidal cells (dark tones) and PV⁺ interneurons (light tones) across WT (black) and Lgi1⁻/⁻ (red) hippocampi (Pyr WT = 1.00 ± 0.02 vs Pyr Lgi1⁻/⁻ = 0.55 ± 0.01 *p* = 7.90 × 10⁻⁶⁰; PV⁺ IN WT = 1.57 ± 0.12 vs PV⁺ IN Lgi1⁻/⁻ = 1.53 ± 0.09; *p* = 1.00; ANOVA followed by Bonferroni's test for means comparison).

cross-react, in native tissues, with closely homologous Kv₁ isoforms. Due to the significant sequence homology between the target peptide sequence in other Kv₁ family members (Fig. 3A), we verified whether at higher protein concentrations, the antibody can recognize other Kv₁ family members. As shown in Fig. 3E,F the antibody showed cross-reactivity with GST fusion proteins containing C-terminal sequences of Kv₁.₁ and to a lesser extent Kv₁.₃ but not Kv₁.₄.

### 3.3. *Kv₁*-associated proteome in WT versus *Lgi1*⁻/⁻ brain homogenate

The capacity of our antibody to recognize Kv₁.₂ and to a lesser extent Kv₁.₁ allows us to address the consequences of the absence of LGI1 on the modification of the proteome associated with these hetero-multimeric potassium channels. Kv₁α (KCNA1 and 2) subunits as well as other Kvβ1 (KCAB1) and β2 (KCAB2) were equally recovered, except Kv₁.₆ (KCNA6) that was less immunoprecipitated to a lesser extent (−32,8%) from *Lgi1*⁻/⁻ extracts (Table S1, and Supplementary sheet).

#### 3.3.1. *Lost Kv₁* partners in *Lgi1*⁻/⁻ samples

The most striking observation was that the landscape of the Kv₁ links with the cytoskeleton showed major rearrangements (Supplementary Tables S2, S3, Supplementary Fig. S2 and Supplementary sheet). The association with ACTC Actin (alpha cardiac muscle 1), the most abundant Kv₁ partner from WT extracts was lost in the *Lgi1*⁻/⁻ samples. This actin isoform was shown to be widely expressed in the hippocampus and other brain structures (Magdaleno et al., 2006). In addition to actin, the co-immunoprecipitation of myosin 10 and the intermediate filament vimentin as well as the actin remodelling protein CYFP2 (cytoplasmic FMR1 Interacting Protein 2 also known as PIR12) (Zhang et al., 2019) (Chen et al., 2014) were completely disrupted in *Lgi1*⁻/⁻. Interestingly, CYFP2 is linked to mTORC signalling (Manigandan and Yun, 2022) and was shown to be involved in epileptic encephalopathy and intellectual disability (Lee et al., 2020). The perturbations in membrane expression and cytoskeletal links of Kv₁ are also reflected by the loss of interaction of Kv₁ with the following partners: i- the membrane-associated
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Kv₁.₂ expression is reduced in the *Lgi1*⁻/⁻ mice.</figcaption></figure>

(A) Quantification by Western blot of Kv₁.₂ expression in brain homogenates of WT (1.00 ± 0.06) and Lgi1⁻/⁻ (0.52 ± 0.04), *p* = 1.03 × 10⁻⁷; *n* = 17; Two-sample t-test. (B) Quantification by Western blot of Kv₁.₂ in in hippocampal homogenates of WT (1.00 ± 0.04) and Lgi1⁻/⁻ (0.38 ± 0.02); *p* = 1.73 × 10⁻¹³; *n* = 20; Two-sample t-test. In a and b, signals were normalized to GAPDH expression levels. (C) representative images of Kv₁.₂ immunoreactivity (IR) across CA1 (top panels, Scale bar = 50 µm), CA3 (middle panels, Scale bar = 50 µm), and DG (bottom panels, Scale bar = 50 µm) in WT vs Lgi1⁻/⁻ mouse. SO: Str. Oriens; SP: Str. Pyramidale; SR: Str. Radiatum; SL: Str. Lucidum; ML: Mol. Layer; GCL: Granule Cell Layer; Hi: Hilus. (D) Quantification of the differences in the normalized Kv₁.₂ IR (to WT levels) through the different hippocampal strata, all comparisons are ***p* < 0.01 (CA1-SO WT = 1.00 ± 0.04 vs Lgi1⁻/⁻ = 0.52 ± 0.02 *p* = 1.58 × 10⁻¹³; CA1-SR WT = 1.00 ± 0.06 vs Lgi1⁻/⁻ = 0.46 ± 0.02 *p* = 8.37 × 10⁻¹⁰; CA3-SO WT = 1.00 ± 0.06 vs Lgi1⁻/⁻ = 0.38 ± 0.02 *p* = 6.32 × 10⁻¹¹; CA3-SL WT = 1.00 ± 0.04 vs Lgi1⁻/⁻ = 0.53 ± 0.03 *p* = 9.61 × 10⁻¹³; CA3-SR WT = 1.00 ± 0.06 vs Lgi1⁻/⁻ = 0.34 ± 0.02 *p* = 7.49 × 10⁻¹¹; DG-ML-SPB WT = 1.00 ± 0.05 vs Lgi1⁻/⁻ = 0.49 ± 0.02 *p* = 1.92 × 10⁻¹¹; DG-Hilus WT = 1.00 ± 0.05 vs Lgi1⁻/⁻ = 0.37 ± 0.01 *p* = 4.15 × 10⁻¹²; DG-ML-IPB WT = 1.00 ± 0.05 vs Lgi1⁻/⁻ = 0.46 ± 0.02 *p* = 1.83 × 10⁻¹⁰; Two-sample t-test. (E) Left, 63× images of Kv1.2 immunoreactivity at CA3 Axonal Initial segments in WT (top panels) and Lgi1⁻/⁻ (bottom panels). Scale bar = 10 µm. Right, detail of a CA3 pyramidal neuron AIS in WT (top panels) and Lgi1⁻/⁻ (bottom panels). Scale bar = 2.5 µm. (F) 63× images showing no differences in Kv₁.₂ IR at the AIS of PV⁺ interneurons. Scale bar = 10 µm (G) Cumulative probability plots of Kv₁.₂ immunoreactivity at individual AIS of WT (black squares) and Lgi1⁻/⁻ (red circles) CA3 pyramidal neurons (WT Pyr CA3 neurons = 1.00 ± 0.01 vs Lgi1⁻/⁻ CA3 Pyr neurons = 0.41 ± 0.01; *p* = 0.00, Kolmogorov-Smirnov Test). (H) Comparisons of Kv₁.₂ immunoreactivity (Normalized to WT CA3 Pyr Neurons) at the AIS of pyramidal cells (dark tones) and PV⁺ interneurons (light tones) across WT (black) and Lgi1⁻/⁻ (red) hippocampi (Pyr WT = 1.00 ± 0.02 vs Pyr Lgi1⁻/⁻ = 0.55 ± 0.01 *p* = 7.90 × 10⁻⁶⁰; PV⁺ IN WT = 1.57 ± 0.12 vs PV⁺ IN Lgi1⁻/⁻ = 1.53 ± 0.09; *p* = 1.00; ANOVA followed by Bonferroni's test for means comparison).

cross-react, in native tissues, with closely homologous Kv₁ isoforms. Due to the significant sequence homology between the target peptide sequence in other Kv₁ family members (Fig. 3A), we verified whether at higher protein concentrations, the antibody can recognize other Kv₁ family members. As shown in Fig. 3E,F the antibody showed cross-reactivity with GST fusion proteins containing C-terminal sequences of Kv₁.₁ and to a lesser extent Kv₁.₃ but not Kv₁.₄.

### 3.3. *Kv₁-associated proteome in WT versus Lgi1⁻/⁻ brain homogenate*

The capacity of our antibody to recognize Kv₁.₂ and to a lesser extent Kv₁.₁ allows us to address the consequences of the absence of LGI1 on the modification of the proteome associated with these hetero-multimeric potassium channels. Kv₁α (KCNA1 and 2) subunits as well as other Kvβ1 (KCAB1) and β2 (KCAB2) were equally recovered, except Kv₁.₆ (KCNA6) that was less immunoprecipitated to a lesser extent (−32,8%) from Lgi1⁻/⁻ extracts (Table S1, and Supplementary sheet).

#### 3.3.1. *Lost Kv₁ partners in Lgi1⁻/⁻ samples*

The most striking observation was that the landscape of the Kv₁ links with the cytoskeleton showed major rearrangements (Supplementary Tables S2, S3, Supplementary Fig. S2 and Supplementary sheet). The association with ACTC Actin (alpha cardiac muscle 1), the most abundant Kv₁ partner from WT extracts was lost in the Lgi1⁻/⁻ samples. This actin isoform was shown to be widely expressed in the hippocampus and other brain structures (Magdaleno et al., 2006). In addition to actin, the co-immunoprecipitation of myosin 10 and the intermediate filament vimentin as well as the actin remodelling protein CYFP2 (cytoplasmic FMR1 Interacting Protein 2 also known as PIR12) (Zhang et al., 2019) (Chen et al., 2014) were completely disrupted in Lgi1⁻/⁻. Interestingly, CYFP2 is linked to mTORC signalling (Manigandan and Yun, 2022) and was shown to be involved in epileptic encephalopathy and intellectual disability (Lee et al., 2020). The perturbations in membrane expression and cytoskeletal links of Kv₁ are also reflected by the loss of interaction of Kv₁ with the following partners: i- the membrane-associated
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Characterization of the rabbit polyclonal Kv₁.₂ antibody.</figcaption></figure>

Isoform specificity of the rabbit polyclonal Kv₁.₂ antibody. (A) Alignment of the amino acid sequences of C-terminal tails of mice Kv₁.₁, Kv₁.₂, Kv₁.₃, Kv₁.₄. The peptide sequence that served to generate the antibody is boxed. (B) HEK cell extracts transfected with recombinant Kv₁.₁ and Kv₁.₂ as well as brain homogenates were probed with the polyclonal Kv₁.₂ antibody. Control recombinant channels expression were performed using the Neuromab Kv₁.₁ and Kv₁.₂ antibodies. Isolated Western blot strips are from distinct gels (C) Western blots of wild type and Kv₁.₂ KO (Kv₁.₁²) brain homogenates with the rabbit polyclonal Kv₁.₂ antibody showing cross-reactivity with closely related native Kv₁ channels. (D) Coomassie staining of the different GST fusion protein constructs (3 µg) used for western blot. (E) and (F) Western blot (300 ng/well) and ELISA (0.5 µg/well) respectively of the constructs shown in b with the polyclonal Kv₁.₂ antibody showing a cross-reactivity of this antibody with Kv₁.₁ and to very slight extent with Kv₁.₃.

phosphatidylinositol (4,5)-bisphosphate binding protein GSDMA, that is
involved in cell death and has pore forming properties (Ding et al.,
2016), ii-the deltex E3 ubiquitin-protein ligase and regulator of Notch
signalling DTX1, iii- the multi-modular scaffolding protein RANB9 that
is implicated in actin cytoskeletal rearrangement (Valiyaveettil et al.,
2008) and the integration of a variety of cell surface receptor signalling
through intracellular targets (Murrin and Talbot, 2007), iv- The plasma
membrane /endoplasmic reticulum (PM/ER) cholesterol transporter
and phosphatidylserine binding protein (Sandhu et al., 2018) ASTRA

that may indicate a change in the lipidic environment of Kv₁. Although a
similar function was attributed to ASTRA and B, the decrease of inter-
action with Kv₁ was restricted to ASTRA, ASTRB remaining stable, v- the
NEB2 (spinophilin) (Molina-Pinelo et al., 2011), a PDZ-domain con-
taining F-actin binding and phosphatase recruiter scaffolding protein.

Intriguingly, a loss of association with PSD93 but not with its twin
PSD95 or other MAGUKs was observed in Lgi1⁻/⁻. We therefore inves-
tigated by Western blot and immunofluorescence whether the loss of
association with PSD93 is accompanied by a decrease in its total
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Characterization of the rabbit polyclonal Kv₁.₂ antibody.</figcaption></figure>

Isoform specificity of the rabbit polyclonal Kv₁.₂ antibody. (A) Alignment of the amino acid sequences of C-terminal tails of mice Kv₁.₁, Kv₁.₂, Kv₁.₃, Kv₁.₄. The peptide sequence that served to generate the antibody is boxed. (B) HEK cell extracts transfected with recombinant Kv₁.₁ and Kv₁.₂ as well as brain homogenates were probed with the polyclonal Kv₁.₂ antibody. Control recombinant channels expression were performed using the Neuromab Kv₁.₁ and Kv₁.₂ antibodies. Isolated Western blot strips are from distinct gels (C) Western blots of wild type and Kv₁.₂ KO (Kv₁.₁²) brain homogenates with the rabbit polyclonal Kv₁.₂ antibody showing cross-reactivity with closely related native Kv₁ channels. (D) Coomassie staining of the different GST fusion protein constructs (3 µg) used for western blot. (E) and (F) Western blot (300 ng/well) and ELISA (0.5 µg/well) respectively of the constructs shown in b with the polyclonal Kv₁.₂ antibody showing a cross-reactivity of this antibody with Kv₁.₁ and to very slight extent with Kv₁.₃.

phosphatidylinositol (4,5)-bisphosphate binding protein GSDMA, that is
involved in cell death and has pore forming properties (Ding et al.,
2016), ii-the deltex E3 ubiquitin-protein ligase and regulator of Notch
signalling DTX1, iii- the multi-modular scaffolding protein RANB9 that
is implicated in actin cytoskeletal rearrangement (Valiyaveettil et al.,
2008) and the integration of a variety of cell surface receptor signalling
through intracellular targets (Murrin and Talbot, 2007), iv- The plasma
membrane /endoplasmic reticulum (PM/ER) cholesterol transporter
and phosphatidylserine binding protein (Sandhu et al., 2018) ASTRA

that may indicate a change in the lipidic environment of Kv₁. Although a
similar function was attributed to ASTRA and B, the decrease of inter-
action with Kv₁ was restricted to ASTRA, ASTRB remaining stable, v- the
NEB2 (spinophilin) (Molina-Pinelo et al., 2011), a PDZ-domain con-
taining F-actin binding and phosphatase recruiter scaffolding protein.

Intriguingly, a loss of association with PSD93 but not with its twin
PSD95 or other MAGUKs was observed in Lgi1⁻/-. We therefore inves-
tigated by Western blot and immunofluorescence whether the loss of
association with PSD93 is accompanied by a decrease in its total
<figure><img src="image_7.png" /><figcaption>Fig. 4. PSD93 expression is reduced in the *Lgl1*<sup>-/-</sup> mice.</figcaption></figure>

(A) Quantification by Western blot of PSD93 expression in brain homogenates of PSD93 in WT (1.00 ± 0.03) and *Lgl1*<sup>-/-</sup> (0.46 ± 0.04); *p* = 1.40 × 10<sup>-11</sup>; *n* = 20; Two-sample *t*-test). (B) Quantification by Western blot of PSD93 expression in hippocampal homogenates in WT (1.00 ± 0.05) and *Lgl1*<sup>-/-</sup> (0.68 ± 0.05); *p* = 6.10 × 10<sup>-5</sup>; *n* = 20; Two-sample *t*-test). In A and B, signals were normalized to GAPDH expression levels. (C) 25× representative images of Hippocampal CA3 region showing a decrease in PSD93 expression. PSD93 is in green, AnkyrinG is in red, and DAPI is in blue. Scale bar = 25 µm. (D) Quantification of PSD93 expression decrease across different CA3 regions in WT (black tones) and *Lgl1*<sup>-/-</sup> (red tones) normalized to WT-SL (CA3-SR WT = 0.85 ± 0.02 vs *Lgl1*<sup>-/-</sup> = 0.51 ± 0.01; *p* = 9.65 × 10<sup>-34</sup>; CA3-SR WT = 1.00 ± 0.02 vs *Lgl1*<sup>-/-</sup> = 0.50 ± 0.01; *p* = 1.31 × 10<sup>-63</sup>; ANOVA followed by Bonferroni's test for means comparison). (E) 63× representative images showing a reduction in PSD93 expression at the AIS level of CA3 Pyramidal neurons in *Lgl1*<sup>-/-</sup>; PSD93 is in green and AnkyrinG is in red; Scale bar = 5 µm. (F) Quantification of the decreased PSD93 expression at the AIS level in *Lgl1*<sup>-/-</sup> hippocampal CA3 pyramidal neurons (WT = WT 1.00 ± 0.03 vs Kv1.1 *Lgl1*<sup>-/-</sup> 0.42 ± 0.02, *p* = 0; Mann-Whitney *U* test). (G) Surface representation of PSD93 labelling at the AIS of CA3 pyramidal neurons in WT (top panels) and *Lgl1*<sup>-/-</sup> (bottom panels) hippocampi. Magenta: AnkG, Green: PSD93.

expression level. As shown in Fig. 4, PSD93 expression levels were severely downregulated in *Lgl1*<sup>-/-</sup> samples with a more prominent decrease in total brain homogenates (PSD93 WT 1.00 ± 0.03 vs PSD93 *Lgl1*<sup>-/-</sup> 0.46 ± 0.04, *p* = 1.40 × 10<sup>-11</sup>) (Fig. 4A) compared to hippocampal extracts (PSD93 WT 1.00 ± 0.05 vs PSD93 *Lgl1*<sup>-/-</sup> 0.68 ± 0.05, *p* = 6.10 × 10<sup>-5</sup>) (Fig. 4B). A closer look at the subcellular distribution of PSD93 indicates that this decrease was not restricted to the AIS (Fig. 4E, F) and was observed also in the stratum radiatum (SR) and stratum lucidum (SL) of CA3 (Fig. 4C, D). Finally, the association of Kv₁ with the myelin proteolipid protein (MYPR), a major determinant in the formation and maintenance of the multilamellar structure of myelin was also lost in *Lgl1*<sup>-/-</sup>. All these molecular signatures indicate a drastic change in Kv₁ membrane and sub-membrane associated complexes and corroborate the significant downregulation of membranous Kv₁ (Seagar et al., 2017).

### 3.3.2. *New Kv₁ partners in Lgl1<sup>-/-</sup> samples*

In parallel to the loss of all the above mentioned membranous, sub-membranous and cytoskeletal links of Kv₁, we found that, in *Lgl1*<sup>-/-</sup> (Supplementary Table S3), Kv₁ engaged novel molecular interactions and showed association with the microtubule associated protein doublecortin (DCX) and with the GTPases Septin 6 and 7 that are involved in actin and microtubule cytoskeleton organisation, membrane trafficking, vesicle transport and exocytosis, as well as the assembly of scaffolding platforms (Neubauer and Zieger, 2017) (Spiliotis, 2018). Interestingly, Septin 6 has been shown to be localized at the neck of dendrites (Cho et al., 2011), suggesting a potential reorganisation of Kv₁ localization. In addition, we found that in *Lgl1*<sup>-/-</sup> samples, Kv₁ is associated with the calcium sensor Copine 6 that regulates structural plasticity of spines. Despite the loss of interaction with actin, Kv₁ in *Lgl1*<sup>-/-</sup> acquired association with the filamentous protein and actin-crosslinking MARCS (Myristoylated alanine-rich C-kinase substrate).
<figure><img src="image_7.png" /><figcaption>Fig. 4. PSD93 expression is reduced in the Lgi1<sup>-/-</sup> mice.</figcaption></figure>

(A) Quantification by Western blot of PSD93 expression in brain homogenates of PSD93 in WT (1.00 ± 0.03) and Lgi1<sup>-/-</sup> (0.46 ± 0.04); p = 1.40 × 10<sup>-11</sup>; n = 20; Two-sample t-test). (B) Quantification by Western blot of PSD93 expression in hippocampal homogenates in WT (1.00 ± 0.05) and Lgi1<sup>-/-</sup> (0.68 ± 0.05); p = 6.10 × 10<sup>-5</sup>; n = 20; Two-sample t-test). In A and B, signals were normalized to GAPDH expression levels. (C) 25× representative images of Hippocampal CA3 region showing a decrease in PSD93 expression. PSD93 is in green, AnkyrinG is in red, and DAPI is in blue. Scale bar = 25 µm. (D) Quantification of PSD93 expression decrease across different CA3 regions in WT (black tones) and Lgi1<sup>-/-</sup> (red tones) normalized to WT-SL (CA3-SR WT = 0.85 ± 0.02 vs Lgi1<sup>-/-</sup> = 0.51 ± 0.01; p = 9.65 × 10<sup>-34</sup>; CA3-SR WT = 1.00 ± 0.02 vs Lgi1<sup>-/-</sup> = 0.50 ± 0.01; p = 1.31 × 10<sup>-63</sup>; ANOVA followed by Bonferroni's test for means comparison). (E) 63× representative images showing a reduction in PSD93 expression at the AIS level of CA3 Pyramidal neurons in Lgi1<sup>-/-</sup>; PSD93 is in green and AnkyrinG is in red; Scale bar = 5 µm. (F) Quantification of the decreased PSD93 expression at the AIS level in Lgi1<sup>-/-</sup> hippocampal CA3 pyramidal neurons (WT = WT 1.00 ± 0.03 vs Kv1.1 Lgi1<sup>-/-</sup> 0.42 ± 0.02, p = 0; Mann-Whitney U test). (G) Surface representation of PSD93 labelling at the AIS of CA3 pyramidal neurons in WT (top panels) and Lgi1<sup>-/-</sup> (bottom panels) hippocampi. Magenta: AnkG, Green: PSD93.

expression level. As shown in Fig. 4, PSD93 expression levels were severely downregulated in Lgi1<sup>-/-</sup> samples with a more prominent decrease in total brain homogenates (PSD93 WT 1.00 ± 0.03 vs PSD93 Lgi1<sup>-/-</sup> 0.46 ± 0.04, p = 1.40 × 10<sup>-11</sup>) (Fig. 4A) compared to hippocampal extracts (PSD93 WT 1.00 ± 0.05 vs PSD93 Lgi1<sup>-/-</sup> 0.68 ± 0.05, p = 6.10 × 10<sup>-5</sup>) (Fig. 4B). A closer look at the subcellular distribution of PSD93 indicates that this decrease was not restricted to the AIS (Fig. 4E, F) and was observed also in the stratum radiatum (SR) and stratum lucidum (SL) of CA3 (Fig. 4C, D). Finally, the association of Kv1 with the myelin proteolipid protein (MYPR), a major determinant in the formation and maintenance of the multilamellar structure of myelin was also lost in Lgi1<sup>-/-</sup>. All these molecular signatures indicate a drastic change in Kv1 membrane and sub-membrane associated complexes and corroborate the significant downregulation of membranous Kv1 (Seagar et al., 2017).

3.3.2. *New Kv₁ partners in Lgi1<sup>-/-</sup> samples*

In parallel to the loss of all the above mentioned membranous, sub-membranous and cytoskeletal links of Kv₁, we found that, in Lgi1⁻/⁻ (Supplementary Table S3), Kv₁ engaged novel molecular interactions and showed association with the microtubule associated protein doublecortin (DCX) and with the GTPases Septin 6 and 7 that are involved in actin and microtubule cytoskeleton organisation, membrane trafficking, vesicle transport and exocytosis, as well as the assembly of scaffolding platforms (Neubauer and Zieger, 2017) (Spiliotis, 2018). Interestingly, Septin 6 has been shown to be localized at the neck of dendrites (Cho et al., 2011), suggesting a potential reorganisation of Kv₁ localization. In addition, we found that in Lgi1⁻/⁻ samples, Kv₁ is associated with the calcium sensor Copine 6 that regulates structural plasticity of spines. Despite the loss of interaction with actin, Kv₁ in Lgi1⁻/⁻ acquired association with the filamentous protein and actin-crosslinking MARCS (Myristoylated alanine-rich C-kinase substrate).
### 3.3.3. Significant changes in the association of some Kv₁ partners in Lgi1⁻/⁻ samples

Among Kv₁ partners that were immunoprecipitated from both wild type and Lgi1⁻/⁻ extracts, those that varied less than ±20% were considered stable. ADAM22 (Disintegrin and metalloproteinase domain-containing protein 22) association was by far the most affected partner with over 60% decrease followed by PSD95 > SAP97 (DLG1) > SAP102 (DLG3) with >50%, > 44% and 40% decrease respectively. Although both ADAM22 and ADAM23 co-immunoprecipitated Kv₁.₂ from pre-enriched LGI1-associated complexes (Fukata et al., 2010), and both proteins associate with LGI1 (Ramirez-Franco et al., 2022), ADAM23 was totally absent from our immunoprecipitations from both WT and Lgi1⁻/⁻ samples.

Beside these partners that show decreased association with Kv₁, an important increase in channel association with several key proteins was observed. The biggest increase in interaction (> 127%) was observed for 14-3-3γ (Ywhag). This increase is accompanied by a > 55% augmentation of interaction with 14-3-3ε. The second largest increase (> 77%)

concerned the vesicle-associated membrane protein-associated protein A (VAPA) tether protein but not VAPB that remained rather stable. In parallel to these differences, there was a noticeable increase of association with the cytosol aminopeptidase AMPL and the leucine-rich repeat and transmembrane domain-containing protein 2 (LRTM2), which are involved in axon guidance. It is interesting to note that while the interaction of Kv₁ with the serine/threonine-protein phosphatase 2B catalytic subunit beta isoform (PP2BB) is lost, the one with the alpha isoform (PP2BA) increases by >34%. In parallel to the interaction with Septin 6 and 7 mentioned earlier, an increase of interaction with Septin 11 was observed. Septin 11 (SEP11), is enriched post-synaptically at the neck of dendritic spines at GABAergic synapses (Li et al., 2009). This observation suggests a reorganisation in the dendritic localisation of Kv₁. Among the partners with increased association with Kv₁, we found WD repeat-containing protein 26 (WDR26) which is a G-beta-like negative regulator of MAPK pathway, haploinsufficiency of which induces among other symptoms intellectual disability and seizures (Skraban et al., 2017). Interestingly, the LGI1 EPTP domain shows

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Kv₂.₁ is selectively reduced in hippocampus of Lgi1⁻/⁻ mice.</figcaption></figure>

(A) Quantification by Western blot of Kv₂.₁ expression in brain homogenates of WT (WT 1.00 ± 0.09) and Lgi1⁻/⁻ (1.01 ± 0.11); *p* = 0.97; *n* = 15; Two-sample t-test. (B) Quantification by Western blot of Kv₂.₁ expression in WT (1.00 ± 0.04) and Lgi1⁻/⁻ (0.45 ± 0.04) hippocampal extracts; *p* = 1.00 × 10⁻⁹; *n* = 16; Two-sample t-test. (C) 10× representative images showing a reduced expression of Kv₂.₁ across the hippocampus of Lgi1⁻/⁻ mouse. Scale bar = 100 µm. (D) Representative 63× images showing a reduction of Kv₂.₁ in CA3 Str. Pyramidal of Lgi1⁻/⁻ hippocampus. Scale bar = 5 µm. (E) Analysis of Kv₂.₁ puncta in CA3 in WT (black) and Lgi1⁻/⁻ (red) hippocampi showing a reduction of Kv₂.₁ clusters' density in Lgi1⁻/⁻. (Density of Clusters (#/µm²) WT = 0.036 ± 0.002 Lgi1⁻/⁻ = 0.020 ± 0.002; *p* = 1.53 × 10⁻⁶; Mann-Whitney U test; Surface of clusters (µm²) WT = 3.48 ± 0.09 Lgi1⁻/⁻ = 3.27 ± 0.08; *p* = 0.08; Two sample t-test; Normalized (to WT) immunoreactivity of clusters WT = 1.00 ± 0.01 Lgi1⁻/⁻ = 0.99 ± 0.01; *p* = 0.53; Two sample t-test; Volume of clusters (µm³) WT = 0.16 ± 0.01 Lgi1⁻/⁻ = 0.15 ± 0.01; *p* = 0.07; Two sample t-test). (F) Representative 63× images showing a reduction of Kv₂.₁ at the level of CA3 Pyr. neurons AIS (CA3 Str. Oriens) of Lgi1⁻/⁻ hippocampus. Scale bar = 5 µm. (G) 63 Detail of a CA3 Pyr. neuron AIS in WT (top) and Lgi1⁻/⁻ (bottom) showing a reduced density of Kv₂.₁ clusters at the AIS of Lgi1⁻/⁻ neurons. Scale bar = 2 µm. (H) Analysis of Kv₂.₁ puncta in the AIS of CA3 pyramidal neurons in WT (black) and Lgi1⁻/⁻ (red) showing a reduction of Kv₂.₁ clusters' density and volume at the AIS of CA3 pyramidal neurons in Lgi1⁻/⁻ mouse. (Density of Clusters (#/µm² AIS) WT = 0.96 ± 0.03 Lgi1⁻/⁻ = 0.69 ± 0.03; *p* = 4.58 × 10⁻⁷; Two sample t-test; Surface of clusters (µm²) WT = 6.87 ± 0.41 Lgi1⁻/⁻ = 5.85 ± 0.27; *p* = 0.07; Mann-Whitney U test; Normalized (to WT) immunoreactivity of clusters WT = 1.00 ± 0.02 Lgi1⁻/⁻ = 0.96 ± 0.01; *p* = 0.09; Mann-Whitney U test; Volume of clusters (µm³) WT = 0.23 ± 0.01 Lgi1⁻/⁻ = 0.18 ± 0.01; *p* = 0.046; Mann-Whitney U test).
### 3.3.3. Significant changes in the association of some Kv₁ partners in Lgi1⁻/⁻ samples

Among Kv₁ partners that were immunoprecipitated from both wild type and Lgi1⁻/⁻ extracts, those that varied less than ±20% were considered stable. ADAM22 (Disintegrin and metalloproteinase domain-containing protein 22) association was by far the most affected partner with over 60% decrease followed by PSD95 > SAP97 (DLG1) > SAP102 (DLG3) with >50%, > 44% and 40% decrease respectively. Although both ADAM22 and ADAM23 co-immunoprecipitated Kv₁.₂ from pre-enriched LGI1-associated complexes (Fukata et al., 2010), and both proteins associate with LGI1 (Ramirez-Franco et al., 2022), ADAM23 was totally absent from our immunoprecipitations from both WT and Lgi1⁻/⁻ samples.

Beside these partners that show decreased association with Kv₁, an important increase in channel association with several key proteins was observed. The biggest increase in interaction (> 127%) was observed for 14-3-3γ (Ywhag). This increase is accompanied by a > 55% augmentation of interaction with 14-3-3ε. The second largest increase (> 77%)

concerned the vesicle-associated membrane protein-associated protein A (VAPA) tether protein but not VAPB that remained rather stable. In parallel to these differences, there was a noticeable increase of association with the cytosol aminopeptidase AMPL and the leucine-rich repeat and transmembrane domain-containing protein 2 (LRTM2), which are involved in axon guidance. It is interesting to note that while the interaction of Kv₁ with the serine/threonine-protein phosphatase 2B catalytic subunit beta isoform (PP2BB) is lost, the one with the alpha isoform (PP2BA) increases by >34%. In parallel to the interaction with Septin 6 and 7 mentioned earlier, an increase of interaction with Septin 11 was observed. Septin 11 (SEP11), is enriched post-synaptically at the neck of dendritic spines at GABAergic synapses (Li et al., 2009). This observation suggests a reorganisation in the dendritic localisation of Kv₁. Among the partners with increased association with Kv₁, we found WD repeat-containing protein 26 (WDR26) which is a G-beta-like negative regulator of MAPK pathway, haploinsufficiency of which induces among other symptoms intellectual disability and seizures (Skraban et al., 2017). Interestingly, the LGI1 EPTP domain shows

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Kv₂.₁ is selectively reduced in hippocampus of Lgi1⁻/⁻ mice.</figcaption></figure>

(A) Quantification by Western blot of Kv₂.₁ expression in brain homogenates of WT (WT 1.00 ± 0.09) and Lgi1⁻/⁻ (1.01 ± 0.11); *p* = 0.97; *n* = 15; Two-sample t-test. (B) Quantification by Western blot of Kv₂.₁ expression in WT (1.00 ± 0.04) and Lgi1⁻/⁻ (0.45 ± 0.04) hippocampal extracts; *p* = 1.00 × 10⁻⁹; *n* = 16; Two-sample t-test. (C) 10× representative images showing a reduced expression of Kv₂.₁ across the hippocampus of Lgi1⁻/⁻ mouse. Scale bar = 100 µm. (D) Representative 63× images showing a reduction of Kv₂.₁ in CA3 Str. Pyramidal of Lgi1⁻/⁻ hippocampus. Scale bar = 5 µm. (E) Analysis of Kv₂.₁ puncta in CA3 in WT (black) and Lgi1⁻/⁻ (red) hippocampi showing a reduction of Kv₂.₁ clusters' density in Lgi1⁻/⁻. (Density of Clusters (#/µm²) WT = 0.036 ± 0.002 Lgi1⁻/⁻ = 0.020 ± 0.002; *p* = 1.53 × 10⁻⁶; Mann-Whitney U test; Surface of clusters (µm²) WT = 3.48 ± 0.09 Lgi1⁻/⁻ = 3.27 ± 0.08; *p* = 0.08; Two sample t-test; Normalized (to WT) immunoreactivity of clusters WT = 1.00 ± 0.01 Lgi1⁻/⁻ = 0.99 ± 0.01; *p* = 0.53; Two sample t-test; Volume of clusters (µm³) WT = 0.16 ± 0.01 Lgi1⁻/⁻ = 0.15 ± 0.01; *p* = 0.07; Two sample t-test). (F) Representative 63× images showing a reduction of Kv₂.₁ at the level of CA3 Pyr. neurons AIS (CA3 Str. Oriens) of Lgi1⁻/⁻ hippocampus. Scale bar = 5 µm. (G) 63 Detail of a CA3 Pyr. neuron AIS in WT (top) and Lgi1⁻/⁻ (bottom) showing a reduced density of Kv₂.₁ clusters at the AIS of Lgi1⁻/⁻ neurons. Scale bar = 2 µm. (H) Analysis of Kv₂.₁ puncta in the AIS of CA3 pyramidal neurons in WT (black) and Lgi1⁻/⁻ (red) showing a reduction of Kv₂.₁ clusters' density and volume at the AIS of CA3 pyramidal neurons in Lgi1⁻/⁻ mouse. (Density of Clusters (#/µm² AIS) WT = 0.96 ± 0.03 Lgi1⁻/⁻ = 0.69 ± 0.03; *p* = 4.58 × 10⁻⁷; Two sample t-test; Surface of clusters (µm²) WT = 6.87 ± 0.41 Lgi1⁻/⁻ = 5.85 ± 0.27; *p* = 0.07; Mann-Whitney U test; Normalized (to WT) immunoreactivity of clusters WT = 1.00 ± 0.02 Lgi1⁻/⁻ = 0.96 ± 0.01; *p* = 0.09; Mann-Whitney U test; Volume of clusters (µm³) WT = 0.23 ± 0.01 Lgi1⁻/⁻ = 0.18 ± 0.01; *p* = 0.046; Mann-Whitney U test).
sequence homology with WD40 sequences (Yamagata et al., 2018) and may reflect a particular propensity of Kv1 subunits to associate with some WD40 motif-containing proteins. This finding supports the suggested changes in the phosphorylation-mediated dynamic balance and stability of Kv₁ membrane expression in Lgi1⁻/⁻ (Connors et al., 2008). In parallel to the acquired association with endocytotic markers, the association of Kv₁ with syntaxin1B was also increased.

### 3.4. *Kv₂* expression is decreased in Lgi1⁻/⁻

Homo-tetrameric *Kv₂* delayed-rectifier potassium channels are widely expressed though hetero-oligomers can occur in some cells (Bishop et al., 2015). VAPA/B interact with *Kv₂*₁ and *Kv₂*₂ channel subunits (Johnson et al., 2018) and induce the formation of channel clusters (Bishop et al., 2015). At PM/ER contact sites, *Kv₂* clusters constitute trafficking hubs where membrane protein insertion and retrieval take place (Johnson et al., 2019). As we observed an increase of VAPA association with *Kv₁* in *Lgi1*⁻/⁻ and a loss of *Kv₁* association with the VAPA interaction partner ASTRA, we undertook to investigate whether *Kv₂* expression in *Lgi1*⁻/⁻ was modified. As shown in Fig. 5a and b, *Kv₂*₁ expression was specifically and drastically reduced in hippocampus (*Kv₂*₁ WT 1.00 ± 0.05 vs *Kv₂*₁ *Lgi1*⁻/⁻ 0.45 ± 0.04, *p* = 1.00 × 10⁻⁹) compared to total brain homogenates (*Kv₂*₁ WT 1.00 ± 0.09 vs *Kv₂*₁ *Lgi1*⁻/⁻ 1.01 ± 0.12, *p* = 0.97) where no difference was observed. *Kv₂* channels provide a major component of somatodendritic potassium currents and occur at the neuronal surface either in conducting freely-diffusing forms or non-conducting clusters (Misonou et al., 2005). These channels are involved in the dynamic modulation of excitability during periods of high neuronal activity (Misonou et al., 2005) (Du et al., 2000). In addition, a decrease in *Kv₂*₁ expression leads to an increase in the propensity of neurons to exhibit complex spike bursting (Raus Balind et al., 2019). In order to get more insight into a potential modification in the expression of *Kv₂*₁ in the *Lgi1*⁻/⁻ hippocampal formation, we performed immunohistochemical staining of *Kv₂*₁ in hippocampal slices. As described earlier (Palacio et al., 2017), *Kv₂*₁ was mostly enriched in the CA1 compared to the CA3 (Fig. 5C). We found that in *Lgi1*⁻/⁻, the expression level of *Kv₂*₁ in the CA3 was severely decreased (Fig. 5C, E). A closer look at the distribution of this channel in the CA3 showed also considerably reduced expression in the stratum oriens (SO) and stratum pyramidale (SP) (Fig. 5D, F) and that AIS labelling was severely diminished (Fig. 5F-H). We further analysed the following four distinct staining parameters, either in global staining or at the AIS: i) cluster density, ii) normalized immunoreactivity of the clusters, iii) cluster surface, and iv) cluster volume. As shown in Fig. 5E and Fig. 5H, cluster density was the only staining parameter that showed significant decrease in the global analysis or at the AIS. This fully converges with the previously reported dispersion of *Kv₂*₁ clusters in response to increased neuronal activity (Misonou et al., 2004) (Romer et al., 2016).

### 3.5. Functional correlate of changes in *Kv*₁ & *Kv*₂ expression levels and interactome changes

LGI1 is particularly enriched in the CA3 hippocampal subregions (Herranz-Perez et al., 2010) (Smedfors et al., 2018) (Ramirez-Franco et al., 2022) and plays a crucial role in regulating CA3 pyramidal neurons excitability (Seagar et al., 2017) (Kornau et al., 2020) (Extremet et al., 2022). Moreover, focal CA3 hippocampal atrophy is observed in autoimmune LGI1-mediated limbic encephalitis (Miller et al., 2017). It has also been pointed out that a decrease in somatodendritic D-type potassium currents (Metz et al., 2007) (Golding et al., 1999) (Golding et al., 2001) (Hyun et al., 2015) (Eom et al., 2019) and a weak expression of *Kv₂* channels (Raus Balind et al., 2019) could promote the appearance of bursting patterns in hippocampal neurons. In order to examine LGI1 deficiency-mediated modification in excitability independently from potential artefacts induced by slice culture conditions,

we examined intrinsic excitability of CA3 pyramidal neurons from acute slices. To examine changes in excitability at the CA3 hippocampal level, we performed current clamp recordings from CA3 pyramidal neurons of WT and their *Lgi1*⁻/⁻ littermates (Fig. 6A, B). Similar to organotypic slices, we observed a marked increase of the excitability in CA3 pyramidal neurons from *Lgi1*⁻/⁻ defined by a pronounced decrease in the rheobase (WT = 119 ± 20 pA vs *Lgi1*⁻/⁻ = 45 ± 4 pA; *p* = 5 × 10⁻⁶, Mann-Whitney U test) and a significant reduction of the latency to the first spike at the rheobase current (WT = 482 ± 40 ms vs. *Lgi1*⁻/⁻ = 341 ± 30 ms; *p* = 0.007, Mann-Whitney U test) (Fig. 6C, D, E). Neither the AP half-width (WT = 1.22 ± 0.02 ms vs. *Lgi1*⁻/⁻ = 1.20 ± 0.02 ms; *p* = 0.55, Two samples t-test), nor amplitude (WT = 90.10 ± 0.93 mV vs. *Lgi1*⁻/⁻ = 92.61 ± 1.00 mV; *p* = 0.07, Two samples t-test) were modified (Fig. 6F-H). This is consistent with previous observations showing that the broadening of AP due to the loss or pharmacological inhibition of *Kv*₁.₁ is only noticeable in presynaptic but not in somatic APs (Vivekananda et al., 2017). Interestingly, we observed a proportion of CA3 pyramidal neurons that exhibit spike bursts at the rheobase (Fig. 6I, J). Although this kind of firing pattern has been previously described (Masukawa et al., 1982) (Raus Balind et al., 2019) in WT cells, the proportion of neurons exhibiting bursts was substantially larger in *Lgi1*⁻/⁻ mice compared to their WT littermates (WTbursting cells = 6.67%, [2/30 cells] vs. *Lgi1*⁻/⁻ bursting cells = 48.57%, [17/35 cells]; *p* = 0.0003 Fisher's exact test) (Fig. 6K, L). In order to address bursting propensity and based on previous work that focused on this type of firing pattern in CA3 pyramidal neurons (Raus Balind et al., 2019), we performed a different set of experiments in which neurons were challenged with either 300 pA or 600 pA current injection pulses (0.5 ms). The proportion of cells bursting at 300pA was significantly larger in *Lgi1*⁻/⁻ mice (WT = 12.5% [1/8 cells] vs. *Lgi1*⁻/⁻ 87.5% [7/8 cells]; *p* = 0.01 Fisher's exact test) and virtually all the *Lgi1*⁻/⁻ cells challenged with 600 pA current injections exhibited a bursting pattern (WT = 12.5% [1/8 cells] vs. *Lgi1*⁻/⁻ 100% [8/8 cells]; *p* = 0.001 Fisher's exact test) (Fig. 6M-O).

## 4. Discussion

In this study we combined immunofluorescence and mass spectrometry experiments to address modifications in *Kv*₁ distribution and interactome in *Lgi1*⁻/⁻. It was previously reported that *Kv*₁ channels undergo a severe downregulation in mice lacking LGI1 (Seagar et al., 2017), as well as upon in vivo hippocampal infusion of LGI1 antibodies (Petit-Pedrol et al., 2018). In this study, we performed a detailed immunohistochemical analysis of *Kv*₁.₁ and *Kv*₁.₂ expression at the *Lgi1*⁻/⁻ hippocampus. The use of antigen retrieval techniques, a necessary condition to reveal *Kv*₁ immunoreactivity at the AIS of hippocampal neurons (Lorincz and Nusser, 2008a), allowed an accurate and reliable analysis of modifications of the *Kv*₁ expression pattern at the cellular and subcellular levels. To our knowledge, this is the first time that *Kv*₁ expression at the AIS has been evaluated in fixed brains of *Lgi1*⁻/⁻ mice. In addition to previous observations on *Kv*₁ downregulation at the AIS, we uncovered an important overall decrease in all neuronal compartments. This finding reflects more adequately the massive decrease in *Kv*₁ expression measured in Western blots since the reduction of *Kv*₁ population in the AIS cannot account for the >50% overall decrease. The relatively stable expression of *Kv*₁.₁ and *Kv*₁.₂ in PV⁺ interneurons is consistent with the finding that the electrophysiological properties of PV interneurons were unchanged in *Lgi1*⁻/⁻ (Zhou et al., 2018), and that deleting LGI1 in PV⁺ interneurons did not influence seizure susceptibility (Boillot et al., 2014).

Our results indicate that, the antibody that we used for immunoprecipitation and mass spectrometry analysis can cross react, but only with closely related and significantly homologous *Kv*₁ family members. The absence of recognition of *Kv*₁.₁ expressed in HEK cells may be due to the limited expression level of recombinant channels in eukaryotic cells. Mass spectrometry analysis of *Kv*₁-associated complexes in *Lgi1*⁻/⁻ revealed intricate molecular changes in the *Kv*₁ interactome. The
sequence homology with WD40 sequences (Yamagata et al., 2018) and may reflect a particular propensity of Kv1 subunits to associate with some WD40 motif-containing proteins. This finding supports the suggested changes in the phosphorylation-mediated dynamic balance and stability of Kv₁ membrane expression in Lgi1⁻/⁻ (Connors et al., 2008). In parallel to the acquired association with endocytotic markers, the association of Kv₁ with syntaxin1B was also increased.

### 3.4. *Kv₂* expression is decreased in *Lgi1*⁻/⁻

Homo-tetrameric *Kv₂* delayed-rectifier potassium channels are widely expressed though hetero-oligomers can occur in some cells (Bishop et al., 2015). VAPA/B interact with *Kv₂*₁ and *Kv₂*₂ channel subunits (Johnson et al., 2018) and induce the formation of channel clusters (Bishop et al., 2015). At PM/ER contact sites, *Kv₂* clusters constitute trafficking hubs where membrane protein insertion and retrieval take place (Johnson et al., 2019). As we observed an increase of VAPA association with *Kv₁* in *Lgi1*⁻/⁻ and a loss of *Kv₁* association with the VAPA interaction partner ASTRA, we undertook to investigate whether *Kv₂* expression in *Lgi1*⁻/⁻ was modified. As shown in Fig. 5a and b, *Kv₂*₁ expression was specifically and drastically reduced in hippocampus (*Kv₂*₁ WT 1.00 ± 0.05 vs *Kv₂*₁ *Lgi1*⁻/⁻ 0.45 ± 0.04, *p* = 1.00 × 10⁻⁹) compared to total brain homogenates (*Kv₂*₁ WT 1.00 ± 0.09 vs *Kv₂*₁ *Lgi1*⁻/⁻ 1.01 ± 0.12, *p* = 0.97) where no difference was observed. *Kv₂* channels provide a major component of somatodendritic potassium currents and occur at the neuronal surface either in conducting freely-diffusing forms or non-conducting clusters (Misonou et al., 2005). These channels are involved in the dynamic modulation of excitability during periods of high neuronal activity (Misonou et al., 2005) (Du et al., 2000). In addition, a decrease in *Kv₂*₁ expression leads to an increase in the propensity of neurons to exhibit complex spike bursting (Raus Balind et al., 2019). In order to get more insight into a potential modification in the expression of *Kv₂*₁ in the *Lgi1*⁻/⁻ hippocampal formation, we performed immunohistochemical staining of *Kv₂*₁ in hippocampal slices. As described earlier (Palacio et al., 2017), *Kv₂*₁ was mostly enriched in the CA1 compared to the CA3 (Fig. 5C). We found that in *Lgi1*⁻/⁻, the expression level of *Kv₂*₁ in the CA3 was severely decreased (Fig. 5C, E). A closer look at the distribution of this channel in the CA3 showed also considerably reduced expression in the stratum oriens (SO) and stratum pyramidale (SP) (Fig. 5D, F) and that AIS labelling was severely diminished (Fig. 5F-H). We further analysed the following four distinct staining parameters, either in global staining or at the AIS: i) cluster density, ii) normalized immunoreactivity of the clusters, iii) cluster surface, and iv) cluster volume. As shown in Fig. 5E and Fig. 5H, cluster density was the only staining parameter that showed significant decrease in the global analysis or at the AIS. This fully converges with the previously reported dispersion of *Kv₂*₁ clusters in response to increased neuronal activity (Misonou et al., 2004) (Romer et al., 2016).

### 3.5. Functional correlate of changes in *Kv₁* & *Kv₂* expression levels and interactome changes

LGI1 is particularly enriched in the CA3 hippocampal subregions (Herranz-Perez et al., 2010) (Smedfors et al., 2018) (Ramirez-Franco et al., 2022) and plays a crucial role in regulating CA3 pyramidal neurons excitability (Seagar et al., 2017) (Kornau et al., 2020) (Extremet et al., 2022). Moreover, focal CA3 hippocampal atrophy is observed in autoimmune LGI1-mediated limbic encephalitis (Miller et al., 2017). It has also been pointed out that a decrease in somatodendritic D-type potassium currents (Metz et al., 2007) (Golding et al., 1999) (Golding et al., 2001) (Hyun et al., 2015) (Eom et al., 2019) and a weak expression of *Kv₂* channels (Raus Balind et al., 2019) could promote the appearance of bursting patterns in hippocampal neurons. In order to examine LGI1 deficiency-mediated modification in excitability independently from potential artefacts induced by slice culture conditions,

we examined intrinsic excitability of CA3 pyramidal neurons from acute slices. To examine changes in excitability at the CA3 hippocampal level, we performed current clamp recordings from CA3 pyramidal neurons of WT and their *Lgi1*⁻/⁻ littermates (Fig. 6A, B). Similar to organotypic slices, we observed a marked increase of the excitability in CA3 pyramidal neurons from *Lgi1*⁻/⁻ defined by a pronounced decrease in the rheobase (WT = 119 ± 20 pA vs *Lgi1*⁻/⁻ = 45 ± 4 pA; *p* = 5 × 10⁻⁶, Mann-Whitney U test) and a significant reduction of the latency to the first spike at the rheobase current (WT = 482 ± 40 ms vs. *Lgi1*⁻/⁻ = 341 ± 30 ms; *p* = 0.007, Mann-Whitney U test) (Fig. 6C, D, E). Neither the AP half-width (WT = 1.22 ± 0.02 ms vs. *Lgi1*⁻/⁻ = 1.20 ± 0.02 ms; *p* = 0.55, Two samples t-test), nor amplitude (WT = 90.10 ± 0.93 mV vs. *Lgi1*⁻/⁻ = 92.61 ± 1.00 mV; *p* = 0.07, Two samples t-test) were modified (Fig. 6F-H). This is consistent with previous observations showing that the broadening of AP due to the loss or pharmacological inhibition of *Kv*₁.₁ is only noticeable in presynaptic but not in somatic APs (Vivekananda et al., 2017). Interestingly, we observed a proportion of CA3 pyramidal neurons that exhibit spike bursts at the rheobase (Fig. 6I, J). Although this kind of firing pattern has been previously described (Masukawa et al., 1982) (Raus Balind et al., 2019) in WT cells, the proportion of neurons exhibiting bursts was substantially larger in *Lgi1*⁻/⁻ mice compared to their WT littermates (WTbursting cells = 6.67%, [2/30 cells] vs. *Lgi1*⁻/⁻ bursting cells = 48.57%, [17/35 cells]; *p* = 0.0003 Fisher's exact test) (Fig. 6K, L). In order to address bursting propensity and based on previous work that focused on this type of firing pattern in CA3 pyramidal neurons (Raus Balind et al., 2019), we performed a different set of experiments in which neurons were challenged with either 300 pA or 600 pA current injection pulses (0.5 ms). The proportion of cells bursting at 300pA was significantly larger in *Lgi1*⁻/⁻ mice (WT = 12.5% [1/8 cells] vs. *Lgi1*⁻/⁻ 87.5% [7/8 cells]; *p* = 0.01 Fisher's exact test) and virtually all the *Lgi1*⁻/⁻ cells challenged with 600 pA current injections exhibited a bursting pattern (WT = 12.5% [1/8 cells] vs. *Lgi1*⁻/⁻ 100% [8/8 cells]; *p* = 0.001 Fisher's exact test) (Fig. 6M-O).

## 4. Discussion

In this study we combined immunofluorescence and mass spectrometry experiments to address modifications in *Kv*₁ distribution and interactome in *Lgi1*⁻/⁻. It was previously reported that *Kv*₁ channels undergo a severe downregulation in mice lacking LGI1 (Seagar et al., 2017), as well as upon in vivo hippocampal infusion of LGI1 antibodies (Petit-Pedrol et al., 2018). In this study, we performed a detailed immunohistochemical analysis of *Kv*₁.₁ and *Kv*₁.₂ expression at the *Lgi1*⁻/⁻ hippocampus. The use of antigen retrieval techniques, a necessary condition to reveal *Kv*₁ immunoreactivity at the AIS of hippocampal neurons (Lorincz and Nusser, 2008a), allowed an accurate and reliable analysis of modifications of the *Kv*₁ expression pattern at the cellular and subcellular levels. To our knowledge, this is the first time that *Kv*₁ expression at the AIS has been evaluated in fixed brains of *Lgi1*⁻/⁻ mice. In addition to previous observations on *Kv*₁ downregulation at the AIS, we uncovered an important overall decrease in all neuronal compartments. This finding reflects more adequately the massive decrease in *Kv*₁ expression measured in Western blots since the reduction of *Kv*₁ population in the AIS cannot account for the >50% overall decrease. The relatively stable expression of *Kv*₁.₁ and *Kv*₁.₂ in PV⁺ interneurons is consistent with the finding that the electrophysiological properties of PV interneurons were unchanged in *Lgi1*⁻/⁻ (Zhou et al., 2018), and that deleting LGI1 in PV⁺ interneurons did not influence seizure susceptibility (Boillot et al., 2014).

Our results indicate that, the antibody that we used for immunoprecipitation and mass spectrometry analysis can cross react, but only with closely related and significantly homologous *Kv*₁ family members. The absence of recognition of *Kv*₁.₁ expressed in HEK cells may be due to the limited expression level of recombinant channels in eukaryotic cells. Mass spectrometry analysis of *Kv*₁-associated complexes in *Lgi1*⁻/⁻ revealed intricate molecular changes in the *Kv*₁ interactome. The
A

WT CA3 Pyramidal cells

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I

Simple spike (SS)

<figure><img src="image_17.png" /></figure>

J

Complex Spike Burst (CSB)

<figure><img src="image_18.png" /></figure>

M

<figure><img src="image_19.png" /></figure>

N

<figure><img src="image_20.png" /></figure>

K

WT

<figure><img src="image_21.png" /></figure>

L

<figure><img src="image_22.png" /></figure>

O

<figure><img src="image_23.png" /></figure>

(caption on next page)
**Fig. 6.** Intrinsic excitability and bursting propensity are increased in *Lgi1*⁻/⁻ CA3 Pyramidal neurons. (A) representative traces for WT CA3 pyramidal neurons in response to the indicated current injections. (B) representative traces for *Lgi1*⁻/⁻ CA3 pyramidal neurons in response to the indicated current injections. (C) Average rheobase values for WT (black) and *Lgi1*⁻/⁻ (red) CA3 pyramidal neurons (WT = 116.00 ± 19.35 pA *Lgi1*⁻/⁻ = 44.86 ± 4.00 pA; *p* = 0.001; Two sample t-test). (D) Average latency values to the first spike at the rheobase in WT (black) and *Lgi1*⁻/⁻ (red) CA3 pyramidal neurons (WT = 475.60 ± 39.33 ms *Lgi1*⁻/⁻ = 341.40 ± 29.83 ms; *p* = 0.007; Two sample t-test). (E) Average input-output curves of WT (black) and *Lgi1*⁻/⁻ (red) CA3 pyramidal neurons. (F) Half-width of action potentials in WT (black) and *Lgi1*⁻/⁻ (red) CA3 pyramidal neurons (WT = 1.22 ± 0.02 ms *Lgi1*⁻/⁻ = 1.20 ± 0.02 ms; *p* = 0.55; Two sample t-test). (G) Amplitude of action potentials in WT (black) and *Lgi1*⁻/⁻ (red) CA3 pyramidal neurons (WT = 90.09 ± 0.93 mV *Lgi1*⁻/⁻ = 92.61 ± 1.00 mV; *p* = 0.07; Two sample t-test). (H) representative traces in current-clamp recordings showing the shape of an AP in WT (black) and *Lgi1*⁻/⁻ (red) CA3 Pyramidal neurons. (I) Non-complex spike bursts (single AP, dark brown) and (J) Complex-spike burst (light brown) recorded in current clamp in CA3 pyramidal neurons. (K) proportion of CSB and Non-CSB neurons at the rheobase in WT CA3 pyramidal neurons. (L) Proportion of CSB and Non-CSB neurons at the rheobase in *Lgi1*⁻/⁻ CA3 pyramidal neurons. (M) Representative traces of bursting propensity in WT CA3 pyramidal neurons studied by suprathreshold current injections at 300 pA (top) and 600 pA (bottom) respectively. (N) Representative traces of bursting propensity in *Lgi1*⁻/⁻ CA3 pyramidal neurons studied by suprathreshold current injections at 300 pA (top) and 600 pA (bottom) respectively. (O) The proportion of CA3 pyramidal cells exhibiting a bursting phenotype is increased in the *Lgi1*⁻/⁻ mouse (at 300 pA CSB WT = 12.5% [1/8 cells] vs. CSB *Lgi1*⁻/⁻ 87.5% [7/8 cells]; *p* = 0.01 Fisher's exact test; at 600 pA CSB WT = 12.5% [1/8 cells] vs. CSB *Lgi1*⁻/⁻ 100% [8/8 cells]; *p* = 0.001 Fisher's exact test). For panels A to L WT *n* = 30 cells from 8 animals; KO *n* = 35 cells from 8 animals. For panels M to O WT *n* = 8 cells from 2 animals; KO *n* = 8 cells from 4 animals.

comparison between Kv1 partners in WT and *Lgi1*⁻/⁻ uncovered intriguing findings, suggesting a dynamic interplay of protein interactions. Certain partners were lost in the absence of LGI1, while new partners were acquired, highlighting the remodelling of Kv₁ interaction networks. The most striking reorganisation is the loss of Kv1 interaction with several cytoskeletal elements. These data are corroborated by a previously published study showing an increased detergent solubility of Kv₁ channel in *Lgi1*⁻/⁻ (Fukata et al., 2010). Besides the major impact on the Kv1 association with the cytoskeletal network, the -significant modification in Kv₁ association with 14-3-3 proteins may be an important readout of the perturbations associated with the absence of LGI1. 14-3-3 are homo/hetero multimeric cytosolic phosphoproteins that are highly expressed in brain and implicated in cell signalling pathways. 14-3-3γ mutations have been shown to be associated with several brain disorders such as epileptic encephalopathy (Allen et al., 2013) (Guella et al., 2017) as well as febrile seizures (Ye et al., 2021). Also, 14-3-3γ plays a pivotal role in oligodendrocytic apoptosis and neuroinflammation (Lee et al., 2015). Recently, it was shown that ADAM22 phosphorylation (S832) stabilizes its interaction with 14-3-3 proteins (ε, ξ, θ, γ, β, and η) and protects it from endocytosis (Yokoi et al., 2021). Of note, the loss of interaction of 14-3-3 with non-phosphorylated ADAM22 does not influence ADAM22 association with LGI1, Kv₁.₂ or PSD95. It is therefore tempting to speculate that in *Lgi1*⁻/⁻ mice, a homeostatic neuronal response aiming at stabilizing membrane Kv₁ drives an increase in ADAM22 / 14-3-3 / Kv₁.₂ association. Further investigation of the Kv₁ phospho-proteome is still necessary to explore these suggestions. The absence of ADAM23 association with Kv1 in wild type samples is surprising in the light of the current view of the literature placing ADAM23 as a presynaptic organizer of Kv1 channels. Previous immunoprecipitations using anti-C-terminal Kv₁.₂ antibodies also do not show association with ADAM23 (Ogawa et al., 2010). Whether the absence of ADAM23 is the result of epitope accessibility in Kv₁ / ADAM23 complexes or reflects a real molecular organisation is still to be addressed.

An additional important perturbation was the sharp reduction in the expression level of PSD93 in whole-brain and hippocampal homogenates, with a prominent reduction at the AIS of CA3 pyramidal neurons. Although PSD93 is one of the major constituents of LGI1 (Fukata et al., 2010) (Hossen et al., 2022) and ADAM22-associated complexes (Fukata et al., 2021), it is weakly associated with Kv₁.₂ in whole brain extracts (Ogawa et al., 2010). The observed specific loss of interaction with PSD93 is consistent with the previously published data showing that PSD93 but not PSD95 (Rasband et al., 2002) (Ogawa et al., 2010) nor ADAM22 (Ogawa et al., 2010), is important in Kv₁.₁ and Kv₁.₂ channel localisation and clustering at AIS (Ogawa et al., 2008). The selective decrease in PSD93 expression contrasts with the reported stability of PSD95 expression level in the *Lgi1*⁻/⁻ background (Lovero et al., 2015) and shows that the association of Kv₁ with PSD93 is dependent on the presence of LGI1. Interestingly, RhoA activity has been shown to be

significantly increased in *Lgi1*⁻/⁻ (Thomas et al., 2018). Also, RhoA activity mediates spine morphological and functional plasticity (Kasai et al., 2021) (Woolfrey and Srivastava, 2016) and may therefore be linked to the observed actin cytoskeleton remodelling. Interestingly, PSD93 phosphorylation by RhoA was recently shown to decrease its binding to ADAM22 and to increase PSD93 heterodimerization with PSD95 (Hossen et al., 2022). The only partner of Kv₁ that interacts with PSD95/PSD93 and that is specifically lost in our immunoprecipitations from *Lgi1*⁻/⁻ tissue is the plasma membrane calcium-transporting ATPase 4 (AT2B4 / PMCA4), that was shown to be enriched in lipid rafts (Sepulveda et al., 2006). AT2B4 interaction with PSD95/93 has been shown to increase with neuronal excitability and glutamate release (Lisek et al., 2017). This increase in excitability leads to the activation of NMDA receptors and triggers AT2B4 selective internalization (Pottorf 2nd et al., 2006). In addition to these observations, it is known that the activity of AT2B4 is inhibited by 14-3-3ε (Rimessi et al., 2005) which interacts with and modulates the expression of the LGI1-binding partner ADAM22. Despite that the protein expression level of AT2B4 (PMCA4) did not show any significant difference between WT and *Lgi1*⁻/⁻ samples (Supplementary Fig. S3), the loss of PSD93 co-immunoprecipitation with Kv₁ in *Lgi1*⁻/⁻ may partially reflect the loss of Kv₁ association with AT2B4. Since LGI1 was shown to regulate PSD95-mediated AMPA and NMDA receptor levels in neurons (Lovero et al., 2015), it may be suggested that, in the *Lgi1*⁻/⁻ epileptic model, a hyperexcitability-driven increase of interaction between PSD95 and AT2B4 could be blocked by the expected increase in RhoA-mediated PSD95/PSD93 interaction. Of note, it was recently reported that the truncation of the MAGUK binding domain of ADAM22 induces a massive reduction in hippocampal PSD95 expression levels (Fukata et al., 2021). In line with these observations, the loss of PSD93 co-immunoprecipitation with Kv₁ in *Lgi1*⁻/⁻ may be the result of a RhoA-kinase phosphorylation of PSD93 and a concomitant delocalization of the PSD95/PSD93 complex. Further analysis is still necessary to understand the specificity of the seemingly key disruption of PSD93 association with Kv₁ complexes in *Lgi1*⁻/⁻.

The most abundant partner of Kv₁ found in *Lgi1*⁻/⁻ is the glyceraldehyde-3-phosphate dehydrogenase (G3P) (Supplementary Table S3, Supplementary Fig. S4) which is found with an emPAI (1.64 ± 0.34) close to the emPAI value of actin (2.12 ± 0.0) in wild type tissue (Supplementary Table S2, Supplementary Fig. S4). G3P is a moonlighting protein involved in several regulatory pathways including synaptic transmission and intermembrane trafficking (Sirover, 2021). The second most abundant acquired interaction partner is the fatty-acid binding protein FABP7 which is involved in the uptake of fatty acids and serves as a cellular chaperone of lipophilic molecules. This protein is expressed in astrocytes and oligodendrocyte progenitor cells (OPCs) (Sharifi et al., 2011) (Sharifi et al., 2013) and has been shown to regulate dendritic morphology and excitatory synaptic function of cortical neurons (Ebrahimi et al., 2016). This observation is in line with the potential expression of Kv₁.₂ in astrocytes (Akhtar et al., 1999). Interestingly,
**Fig. 6.** Intrinsic excitability and bursting propensity are increased in *Lgi1*⁻/⁻ CA3 Pyramidal neurons. (A) representative traces for WT CA3 pyramidal neurons in response to the indicated current injections. (B) representative traces for *Lgi1*⁻/⁻ CA3 pyramidal neurons in response to the indicated current injections. (C) Average rheobase values for WT (black) and *Lgi1*⁻/⁻ (red) CA3 pyramidal neurons (WT = 116.00 ± 19.35 pA *Lgi1*⁻/⁻ = 44.86 ± 4.00 pA; *p* = 0.001; Two sample t-test). (D) Average latency values to the first spike at the rheobase in WT (black) and *Lgi1*⁻/⁻ (red) CA3 pyramidal neurons (WT = 475.60 ± 39.33 ms *Lgi1*⁻/⁻ = 341.40 ± 29.83 ms; *p* = 0.007; Two sample t-test). (E) Average input-output curves of WT (black) and *Lgi1*⁻/⁻ (red) CA3 pyramidal neurons. (F) Half-width of action potentials in WT (black) and *Lgi1*⁻/⁻ (red) CA3 pyramidal neurons (WT = 1.22 ± 0.02 ms *Lgi1*⁻/⁻ = 1.20 ± 0.02 ms; *p* = 0.55; Two sample t-test). (G) Amplitude of action potentials in WT (black) and *Lgi1*⁻/⁻ (red) CA3 pyramidal neurons (WT = 90.09 ± 0.93 mV *Lgi1*⁻/⁻ = 92.61 ± 1.00 mV; *p* = 0.07; Two sample t-test). (H) representative traces in current-clamp recordings showing the shape of an AP in WT (black) and *Lgi1*⁻/⁻ (red) CA3 Pyramidal neurons. (I) Non-complex spike bursts (single AP, dark brown) and (J) Complex-spike burst (light brown) recorded in current clamp in CA3 pyramidal neurons. (K) proportion of CSB and Non-CSB neurons at the rheobase in WT CA3 pyramidal neurons. (L) Proportion of CSB and Non-CSB neurons at the rheobase in *Lgi1*⁻/⁻ CA3 pyramidal neurons. (M) Representative traces of bursting propensity in WT CA3 pyramidal neurons studied by suprathreshold current injections at 300 pA (top) and 600 pA (bottom) respectively. (N) Representative traces of bursting propensity in *Lgi1*⁻/⁻ CA3 pyramidal neurons studied by suprathreshold current injections at 300 pA (top) and 600 pA (bottom) respectively. (O) The proportion of CA3 pyramidal cells exhibiting a bursting phenotype is increased in the *Lgi1*⁻/⁻ mouse (at 300 pA CSB WT = 12.5% [1/8 cells] vs. CSB *Lgi1*⁻/⁻ 87.5% [7/8 cells]; *p* = 0.01 Fisher's exact test; at 600 pA CSB WT = 12.5% [1/8 cells] vs. CSB *Lgi1*⁻/⁻ 100% [8/8 cells]; *p* = 0.001 Fisher's exact test). For panels A to L WT *n* = 30 cells from 8 animals; KO *n* = 35 cells from 8 animals. For panels M to O WT *n* = 8 cells from 2 animals; KO *n* = 8 cells from 4 animals.

comparison between Kv1 partners in WT and *Lgi1*⁻/⁻ uncovered intriguing findings, suggesting a dynamic interplay of protein interactions. Certain partners were lost in the absence of LGI1, while new partners were acquired, highlighting the remodelling of Kv₁ interaction networks. The most striking reorganisation is the loss of Kv1 interaction with several cytoskeletal elements. These data are corroborated by a previously published study showing an increased detergent solubility of Kv₁ channel in *Lgi1*⁻/⁻ (Fukata et al., 2010). Besides the major impact on the Kv1 association with the cytoskeletal network, the -significant modification in Kv₁ association with 14-3-3 proteins may be an important readout of the perturbations associated with the absence of LGI1. 14-3-3 are homo/hetero multimeric cytosolic phosphoproteins that are highly expressed in brain and implicated in cell signalling pathways. 14-3-3γ mutations have been shown to be associated with several brain disorders such as epileptic encephalopathy (Allen et al., 2013) (Guella et al., 2017) as well as febrile seizures (Ye et al., 2021). Also, 14-3-3γ plays a pivotal role in oligodendrocytic apoptosis and neuroinflammation (Lee et al., 2015). Recently, it was shown that ADAM22 phosphorylation (S₈₃₂) stabilizes its interaction with 14-3-3 proteins (ε, ξ, θ, γ, β, and η) and protects it from endocytosis (Yokoi et al., 2021). Of note, the loss of interaction of 14-3-3 with non-phosphorylated ADAM22 does not influence ADAM22 association with LGI1, Kv₁.₂ or PSD95. It is therefore tempting to speculate that in *Lgi1*⁻/⁻ mice, a homeostatic neuronal response aiming at stabilizing membrane Kv₁ drives an increase in ADAM22 / 14-3-3 / Kv₁.₂ association. Further investigation of the Kv₁ phospho-proteome is still necessary to explore these suggestions. The absence of ADAM23 association with Kv1 in wild type samples is surprising in the light of the current view of the literature placing ADAM23 as a presynaptic organizer of Kv1 channels. Previous immunoprecipitations using anti-C-terminal Kv₁.₂ antibodies also do not show association with ADAM23 (Ogawa et al., 2010). Whether the absence of ADAM23 is the result of epitope accessibility in Kv₁ / ADAM23 complexes or reflects a real molecular organisation is still to be addressed.

An additional important perturbation was the sharp reduction in the expression level of PSD93 in whole-brain and hippocampal homogenates, with a prominent reduction at the AIS of CA3 pyramidal neurons. Although PSD93 is one of the major constituents of LGI1 (Fukata et al., 2010) (Hossen et al., 2022) and ADAM22-associated complexes (Fukata et al., 2021), it is weakly associated with Kv₁.₂ in whole brain extracts (Ogawa et al., 2010). The observed specific loss of interaction with PSD93 is consistent with the previously published data showing that PSD93 but not PSD95 (Rasband et al., 2002) (Ogawa et al., 2010) nor ADAM22 (Ogawa et al., 2010), is important in Kv₁.₁ and Kv₁.₂ channel localisation and clustering at AIS (Ogawa et al., 2008). The selective decrease in PSD93 expression contrasts with the reported stability of PSD95 expression level in the *Lgi1*⁻/⁻ background (Lovero et al., 2015) and shows that the association of Kv₁ with PSD93 is dependent on the presence of LGI1. Interestingly, RhoA activity has been shown to be

significantly increased in *Lgi1*⁻/⁻ (Thomas et al., 2018). Also, RhoA activity mediates spine morphological and functional plasticity (Kasai et al., 2021) (Woolfrey and Srivastava, 2016) and may therefore be linked to the observed actin cytoskeleton remodelling. Interestingly, PSD93 phosphorylation by RhoA was recently shown to decrease its binding to ADAM22 and to increase PSD93 heterodimerization with PSD95 (Hossen et al., 2022). The only partner of Kv₁ that interacts with PSD95/PSD93 and that is specifically lost in our immunoprecipitations from *Lgi1*⁻/⁻ tissue is the plasma membrane calcium-transporting ATPase 4 (AT2B4 / PMCA4), that was shown to be enriched in lipid rafts (Sepulveda et al., 2006). AT2B4 interaction with PSD95/93 has been shown to increase with neuronal excitability and glutamate release (Lisek et al., 2017). This increase in excitability leads to the activation of NMDA receptors and triggers AT2B4 selective internalization (Pottorf 2nd et al., 2006). In addition to these observations, it is known that the activity of AT2B4 is inhibited by 14-3-3ε (Rimessi et al., 2005) which interacts with and modulates the expression of the LGI1-binding partner ADAM22. Despite that the protein expression level of AT2B4 (PMCA4) did not show any significant difference between WT and *Lgi1*⁻/⁻ samples (Supplementary Fig. S3), the loss of PSD93 co-immunoprecipitation with Kv₁ in *Lgi1*⁻/⁻ may partially reflect the loss of Kv₁ association with AT2B4. Since LGI1 was shown to regulate PSD95-mediated AMPA and NMDA receptor levels in neurons (Lovero et al., 2015), it may be suggested that, in the *Lgi1*⁻/⁻ epileptic model, a hyperexcitability-driven increase of interaction between PSD95 and AT2B4 could be blocked by the expected increase in RhoA-mediated PSD95/PSD93 interaction. Of note, it was recently reported that the truncation of the MAGUK binding domain of ADAM22 induces a massive reduction in hippocampal PSD95 expression levels (Fukata et al., 2021). In line with these observations, the loss of PSD93 co-immunoprecipitation with Kv₁ in *Lgi1*⁻/⁻ may be the result of a RhoA-kinase phosphorylation of PSD93 and a concomitant delocalization of the PSD95/PSD93 complex. Further analysis is still necessary to understand the specificity of the seemingly key disruption of PSD93 association with Kv₁ complexes in *Lgi1*⁻/⁻.

The most abundant partner of Kv₁ found in *Lgi1*⁻/⁻ is the glyceraldehyde-3-phosphate dehydrogenase (G3P) (Supplementary Table S3, Supplementary Fig. S4) which is found with an emPAI (1.64 ± 0.34) close to the emPAI value of actin (2.12 ± 0.0) in wild type tissue (Supplementary Table S2, Supplementary Fig. S4). G3P is a moonlighting protein involved in several regulatory pathways including synaptic transmission and intermembrane trafficking (Sirover, 2021). The second most abundant acquired interaction partner is the fatty-acid binding protein FABP7 which is involved in the uptake of fatty acids and serves as a cellular chaperone of lipophilic molecules. This protein is expressed in astrocytes and oligodendrocyte progenitor cells (OPCs) (Sharifi et al., 2011) (Sharifi et al., 2013) and has been shown to regulate dendritic morphology and excitatory synaptic function of cortical neurons (Ebrahimi et al., 2016). This observation is in line with the potential expression of Kv₁.₂ in astrocytes (Akhtar et al., 1999). Interestingly,
among the newly acquired partners we found PCMD2 (protein-L-iso-aspartate O-methyltransferase domain-containing protein 2) a member of the L-isoaspartyl O-methyltransferases (PIMT) protein family, known to repair natural protein damage (Kim et al., 1997) (Yamamoto et al., 1998) (Reissner et al., 2006) (Zhu et al., 2006). The presence of this protein in Kv1-associated complexes may suggest that at least some of its components may be under repair and imply a higher level of protein damage/recycling than in WT tissue. The presence of synapsin 2 (SYN2) and the voltage-dependent anion-selective channel protein 1 (VDAC1) in the Kv₁ interactome of Lgi1⁻/⁻ corroborates this hypothesis since these proteins have been identified as major endogenous substrates for PIMT (Zhu et al., 2006). The association with the ATPase-dependent chaperone T-complex protein 1 subunit delta (TCPD) is in line with this interpretation and may highlight attempts for recovery from damage. This protein localises to dendrites where it is involved in microtubule organisation (Wang et al., 2020).

Trafficking pathways associated with Kv₁ also seem to have been affected in Lgi1⁻/⁻ tissue, as the presence of AP1- and AP3-associated coat protein PACS1 decreased by >29% and the association with PSD1 (Cytohesin 1), a guanine nucleotide exchange factor for ARF6 involved in neuron projection development, protein trafficking and cytoskeletal rearrangements (Toma-Fukai and Shimizu, 2019), decreased by >38%. The acquisition of Kv1 association with VAMP2, syntaxin1A, synaptogyrin 3, copine1, synapsin as well as dynamin 3 (DYN3) indicate the presence of Kv₁ in vesicular compartments and suggest an increase in channel endocytosis. This is corroborated by the fact that neuronal activity blockade was shown to induce synaptic clustering of post-synaptic DYN3 (Calabrese and Halpain, 2015) (Gray et al., 2003) (Gray et al., 2005) and that TTX-induced neuronal activity blockade led to a reduction of the Kv1-mediated DTX-I sensitive current (Cudmore et al., 2010). Also, it has been shown that activity-dependent Kv₁.₂ endocytosis in distal apical dendrites is mediated by DYN3 in CA3 pyramidal neurons (Hyun et al., 2013). Therefore, the acquirement of DYN3 association with Kv₁ may be the read out of this mechanism. Since Kv1 levels are reduced in Lgi1⁻/⁻, the association of Kv₁ with DYN3 might reflect a mechanism of Kv₁ retrieval at the dendritic level. This hypothesis is corroborated by the acquired Kv₁ association in Lgi1⁻/⁻ with annexin A2 (ANXA2), which is a membrane-associated pleiotropic calcium-dependent phospholipid binding protein involved in several aspects of cell biology related to calcium signalling and endocytosis. ANXA2 is enriched in lipid rafts (Zhao et al., 2004) and its secretion is activity-dependent (Zhao and Lu, 2007). It has been shown to be primarily present in GABAergic interneurons (Zhao and Lu, 2007) and is overexpressed in pathological neuronal and glial reactions (Eberhard et al., 1994). Therefore, its presence in the Kv₁ interactome in an Lgi1⁻/⁻ background may be linked to the observed astrogliosis in these animals.

In addition to the perturbation of Kv₁ cytoskeletal links, Kv₁ transit from ER/Golgi to plasma membranes seems also to be affected since the association of Kv₁ with the Golgi adaptors AP3S1 and AP1B1 (Nakatsu et al., 2014) is disrupted. These adaptor proteins are known to play a crucial role in sorting the cargo destined to plasma membranes, neurites and nerve terminals (Larimore et al., 2011) (Clemens Grisham et al., 2013).

An ER population of Kv₁ seems to be particularly prominent in Lgi1⁻/⁻ samples. This conclusion is driven by the fact that in Lgi1⁻/⁻, Kv₁ is associated with LRRC59 and RPN1. LRRC59 is an ER mRNA receptor known to participate in coupling translation and ER membrane insertion of transmembrane-domain containing proteins (Bhadra et al., 2021) and RPN1 (dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 is involved in early glycosylation steps of nascent proteins. The association with the receptor of activated protein C kinase 1 (RACK1), PKCγ (KPCG), the cAMP-dependent protein kinase catalytic subunit beta (KAPCB) and the adenylate cyclase activator LANC2 (Fresia et al., 2016) may reflect a change in phosphorylation-mediated dynamic balance and stability of Kv₁ membrane expression (Connors et al., 2008). Finally, the acquisition of association with L-lactate dehydrogenase B chain (LDHB)

may indicate a functional modulation of channel activity since LDBH is part of the oxydoreductase complex and is involved in NAD metabolism which is important in Kv₁.₂ activity modulation (Vacher and Trimmer, 2011). In parallel to this association, Kv₁ coimmunoprecipitated with the voltage-dependent anion-selective channel protein 1 (VDAC1) that binds sphingolipids, cholesterol and phosphatidylcholine and is involved in apoptosis and energy metabolism.

All these results underscore the complex nature of Kv₁ interactions, where the absence of LGI1 leads to an elaborate rewiring of protein partnerships, resulting in both gains and losses that intricately modulate the network dynamics and underly a complex deleterious phenotype in the Lgi1⁻/⁻ mouse.

In parallel to the direct observations driven by mass spectrometry analysis, we report for the first time a massive hippocampal reduction in Kv₂ in Lgi1⁻/⁻. Similar to AMPA receptors that are downregulated in Lgi1⁻/⁻ but are not immonuprecipitated with LGI1 antibodies, Kv₂ is downregulated in Lgi1⁻/⁻ but was not immunoprecipitated with Kv₁ antibodies. This suggests that in Lgi1⁻/⁻, downregulation of AMPA receptors and Kv₂ channels may be the result of a complex molecular reorganisation in response to increased neuronal excitability. As a matter of fact, in Lgi1⁻/⁻ the interaction of Kv₁ with the F-actin-binding and phosphatase recruiter NEB2 is lost. This protein is a regulatory subunit of protein phosphatase 1 (Molina-Pinelo et al., 2011) that controls serine/threonine dephosphorylation of several membrane proteins such as ion channels and certain G-protein coupled receptors (Misonou et al., 2004) and is enriched in postsynaptic compartments (Satoh et al., 1998) where it is involved in modulating spine maturation and morphology (Wang et al., 2021). In accordance with this observation, PP2BB association with Kv₁ was lost in Lgi1⁻/⁻. PP2BB is a regulatory subunit of the activity-dependent serine/threonine-protein phosphatase calcineurin and plays an essential role in intracellular Ca²⁺-mediated signal transduction. With PP1, it plays a regulatory role in Kv₂.₁ phosphorylation modulating its activity, membrane clustering and internalization with key effects on the control of neuronal excitability (Misonou et al., 2006) (Misonou et al., 2004). Interestingly, the interaction of NEB2 with actin is modulated by doublecortin (DCX) (Tsukada et al., 2005) and we found that this protein is acquired as a new partner of Kv₁ in Lgi1⁻/⁻ samples. Also, the interaction with Kv₁ of the tether protein VAPA was massively upregulated in Lgi1⁻/⁻. VAPA/B are Kv₂-interacting tether proteins located at membrane contact sites between the endoplasmic reticulum (ER) and the plasma membrane (PM) (James and Kehlenbach, 2021) which are rich in AKAPs, kinases, Rabs and non-vesicular lipid transfer proteins (i.e ASTRA, ASTRB (Ferrari et al., 2020)). VAPA/B possess a large number of common interactors, although, a specific interactome for each VAP does exist, it does not point to any clear functional difference between the two (James and Kehlenbach, 2021). The reason for the selective change in VAPA remains obscure, however, it may be related to the observed increase in the ER population of Kv₁ and suggests that LGI1 is involved in the presence of Kv₁ channels at ER/PM contact sites. Whether the increase in VAPA levels is linked to the increase in Kv₁ association with ER proteins needs further investigation.

In addition to the crucial role of Kv₁ channels in tuning the excitability of CA3 pyramidal neurons (Rama et al., 2017), its decrease at the somatodendritic compartment may trigger an increased afterdepolarization (Metz et al., 2007). Moreover, the reduction in Kv₂ expression may trigger an increase in dendritic calcium spikes. These two different mechanisms could act synergistically facilitating bursting (Metz et al., 2007) (Raus Balind et al., 2019). Our results are also in agreement with other described forms of increased excitability in Lgi1⁻/⁻ mice (Yu et al., 2010) (Zhou et al., 2018) (Boillot et al., 2016), and with the observed increase in excitability upon the treatment of slices with LGI1 antibodies (Lalic et al., 2011) (Kornau et al., 2020) (Extremet et al., 2022). The importance of Kv₂ downregulation in perturbing excitability in LGI1 deficiency is also highlighted by the fact that restoring Kv₁.₁ expression in secretion-deficient LGI1W183R neurons does
among the newly acquired partners we found PCMD2 (protein-L-iso-aspartate O-methyltransferase domain-containing protein 2) a member of the L-isoaspartyl O-methyltransferases (PIMT) protein family, known to repair natural protein damage (Kim et al., 1997) (Yamamoto et al., 1998) (Reissner et al., 2006) (Zhu et al., 2006). The presence of this protein in Kv1-associated complexes may suggest that at least some of its components may be under repair and imply a higher level of protein damage/recycling than in WT tissue. The presence of synapsin 2 (SYN2) and the voltage-dependent anion-selective channel protein 1 (VDAC1) in the Kv₁ interactome of Lgi1⁻/⁻ corroborates this hypothesis since these proteins have been identified as major endogenous substrates for PIMT (Zhu et al., 2006). The association with the ATPase-dependent chaperone T-complex protein 1 subunit delta (TCPD) is in line with this interpretation and may highlight attempts for recovery from damage. This protein localises to dendrites where it is involved in microtubule organisation (Wang et al., 2020).

Trafficking pathways associated with Kv₁ also seem to have been affected in Lgi1⁻/⁻ tissue, as the presence of AP1- and AP3-associated coat protein PACS1 decreased by >29% and the association with PSD1 (Cytohesin 1), a guanine nucleotide exchange factor for ARF6 involved in neuron projection development, protein trafficking and cytoskeletal rearrangements (Toma-Fukai and Shimizu, 2019), decreased by >38%. The acquisition of Kv1 association with VAMP2, syntaxin1A, synaptogyrin 3, copine1, synapsin as well as dynamin 3 (DYN3) indicate the presence of Kv₁ in vesicular compartments and suggest an increase in channel endocytosis. This is corroborated by the fact that neuronal activity blockade was shown to induce synaptic clustering of post-synaptic DYN3 (Calabrese and Halpain, 2015) (Gray et al., 2003) (Gray et al., 2005) and that TTX-induced neuronal activity blockade led to a reduction of the Kv1-mediated DTX-I sensitive current (Cudmore et al., 2010). Also, it has been shown that activity-dependent Kv₁.₂ endocytosis in distal apical dendrites is mediated by DYN3 in CA3 pyramidal neurons (Hyun et al., 2013). Therefore, the acquirement of DYN3 association with Kv₁ may be the read out of this mechanism. Since Kv1 levels are reduced in Lgi1⁻/⁻, the association of Kv₁ with DYN3 might reflect a mechanism of Kv₁ retrieval at the dendritic level. This hypothesis is corroborated by the acquired Kv₁ association in Lgi1⁻/⁻ with annexin A2 (ANXA2), which is a membrane-associated pleiotropic calcium-dependent phospholipid binding protein involved in several aspects of cell biology related to calcium signalling and endocytosis. ANXA2 is enriched in lipid rafts (Zhao et al., 2004) and its secretion is activity-dependent (Zhao and Lu, 2007). It has been shown to be primarily present in GABAergic interneurons (Zhao and Lu, 2007) and is overexpressed in pathological neuronal and glial reactions (Eberhard et al., 1994). Therefore, its presence in the Kv₁ interactome in an Lgi1⁻/⁻ background may be linked to the observed astrogliosis in these animals.

In addition to the perturbation of Kv₁ cytoskeletal links, Kv₁ transit from ER/Golgi to plasma membranes seems also to be affected since the association of Kv₁ with the Golgi adaptors AP3S1 and AP1B1 (Nakatsu et al., 2014) is disrupted. These adaptor proteins are known to play a crucial role in sorting the cargo destined to plasma membranes, neurites and nerve terminals (Larimore et al., 2011) (Clemens Grisham et al., 2013).

An ER population of Kv₁ seems to be particularly prominent in Lgi1⁻/⁻ samples. This conclusion is driven by the fact that in Lgi1⁻/⁻, Kv₁ is associated with LRRC59 and RPN1. LRRC59 is an ER mRNA receptor known to participate in coupling translation and ER membrane insertion of transmembrane-domain containing proteins (Bhadra et al., 2021) and RPN1 (dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 is involved in early glycosylation steps of nascent proteins. The association with the receptor of activated protein C kinase 1 (RACK1), PKCγ (KPCG), the cAMP-dependent protein kinase catalytic subunit beta (KAPCB) and the adenylate cyclase activator LANC2 (Fresia et al., 2016) may reflect a change in phosphorylation-mediated dynamic balance and stability of Kv₁ membrane expression (Connors et al., 2008). Finally, the acquisition of association with L-lactate dehydrogenase B chain (LDHB)

may indicate a functional modulation of channel activity since LDBH is part of the oxydoreductase complex and is involved in NAD metabolism which is important in Kv₁.₂ activity modulation (Vacher and Trimmer, 2011). In parallel to this association, Kv₁ coimmunoprecipitated with the voltage-dependent anion-selective channel protein 1 (VDAC1) that binds sphingolipids, cholesterol and phosphatidylcholine and is involved in apoptosis and energy metabolism.

All these results underscore the complex nature of Kv₁ interactions, where the absence of LGI1 leads to an elaborate rewiring of protein partnerships, resulting in both gains and losses that intricately modulate the network dynamics and underly a complex deleterious phenotype in the Lgi1⁻/⁻ mouse.

In parallel to the direct observations driven by mass spectrometry analysis, we report for the first time a massive hippocampal reduction in Kv₂ in Lgi1⁻/⁻. Similar to AMPA receptors that are downregulated in Lgi1⁻/⁻ but are not immonuprecipitated with LGI1 antibodies, Kv₂ is downregulated in Lgi1⁻/⁻ but was not immunoprecipitated with Kv₁ antibodies. This suggests that in Lgi1⁻/⁻, downregulation of AMPA receptors and Kv₂ channels may be the result of a complex molecular reorganisation in response to increased neuronal excitability. As a matter of fact, in Lgi1⁻/⁻ the interaction of Kv₁ with the F-actin-binding and phosphatase recruiter NEB2 is lost. This protein is a regulatory subunit of protein phosphatase 1 (Molina-Pinelo et al., 2011) that controls serine/threonine dephosphorylation of several membrane proteins such as ion channels and certain G-protein coupled receptors (Misonou et al., 2004) and is enriched in postsynaptic compartments (Satoh et al., 1998) where it is involved in modulating spine maturation and morphology (Wang et al., 2021). In accordance with this observation, PP2BB association with Kv₁ was lost in Lgi1⁻/⁻. PP2BB is a regulatory subunit of the activity-dependent serine/threonine-protein phosphatase calcineurin and plays an essential role in intracellular Ca²⁺-mediated signal transduction. With PP1, it plays a regulatory role in Kv₂.₁ phosphorylation modulating its activity, membrane clustering and internalization with key effects on the control of neuronal excitability (Misonou et al., 2006) (Misonou et al., 2004). Interestingly, the interaction of NEB2 with actin is modulated by doublecortin (DCX) (Tsukada et al., 2005) and we found that this protein is acquired as a new partner of Kv₁ in Lgi1⁻/⁻ samples. Also, the interaction with Kv₁ of the tether protein VAPA was massively upregulated in Lgi1⁻/⁻. VAPA/B are Kv₂-interacting tether proteins located at membrane contact sites between the endoplasmic reticulum (ER) and the plasma membrane (PM) (James and Kehlenbach, 2021) which are rich in AKAPs, kinases, Rabs and non-vesicular lipid transfer proteins (i.e ASTRA, ASTRB (Ferrari et al., 2020)). VAPA/B possess a large number of common interactors, although, a specific interactome for each VAP does exist, it does not point to any clear functional difference between the two (James and Kehlenbach, 2021). The reason for the selective change in VAPA remains obscure, however, it may be related to the observed increase in the ER population of Kv₁ and suggests that LGI1 is involved in the presence of Kv₁ channels at ER/PM contact sites. Whether the increase in VAPA levels is linked to the increase in Kv₁ association with ER proteins needs further investigation.

In addition to the crucial role of Kv₁ channels in tuning the excitability of CA3 pyramidal neurons (Rama et al., 2017), its decrease at the somatodendritic compartment may trigger an increased afterdepolarization (Metz et al., 2007). Moreover, the reduction in Kv₂ expression may trigger an increase in dendritic calcium spikes. These two different mechanisms could act synergistically facilitating bursting (Metz et al., 2007) (Raus Balind et al., 2019). Our results are also in agreement with other described forms of increased excitability in Lgi1⁻/⁻ mice (Yu et al., 2010) (Zhou et al., 2018) (Boillot et al., 2016), and with the observed increase in excitability upon the treatment of slices with LGI1 antibodies (Lalic et al., 2011) (Kornau et al., 2020) (Extremet et al., 2022). The importance of Kv₂ downregulation in perturbing excitability in LGI1 deficiency is also highlighted by the fact that restoring Kv₁.₁ expression in secretion-deficient LGI1W183R neurons does
not fully rescue the life span of mice (Zhou et al., 2023). Further investigations are needed to address whether LGI1 is directly implicated in modulating Kv₂ expression levels and whether Kv₁ downregulation is parallel to or consequent to Kv₂ downregulation.

In the light of the concomitant decrease in Kv₁ and Kv₂ expression levels, we addressed the intrinsic excitability of CA3 pyramidal neurons of *Lgi1*⁻/⁻. We and others have previously shown that LGI1 is particularly enriched in CA3 hippocampal subregion (Herranz-Perez et al., 2010) (Smedfors et al., 2018) (Ramirez-Franco et al., 2022) and that LGI1 is crucial in controlling neuronal excitability in CA3 pyramidal neurons (Seagar et al., 2017) (Kornau et al., 2020) (Extremet et al., 2022). Moreover, LGI1 antibody-mediated limbic encephalitis is coincident with focal CA3 hippocampal atrophy in human patients (Miller et al., 2017). Although we have previously characterized the intrinsic excitability in pyramidal CA3 neurons of *Lgi1*⁻/⁻ cultured hippocampal slices (Seagar et al., 2017), it has been proposed that cultured hippocampal slices spontaneously develop epileptiform activity (Dyhrfjeld-Johnsen et al., 2010). To eliminate a potential bias introduced by the use of organotypic cultures, and to gain further understanding of the mechanisms underlying LGI1 deficiency-mediated epilepsy, we analysed intrinsic excitability of CA3 pyramidal neurons from acute slices.

We observed an increase in neuronal burst firing propensity and intrinsic excitability defined as a decrease in the rheobase and a shortened latency to the first spike. This gain in excitability converges with the decrease in Kv₁.₁ and Kv₁.₂ as well as Kv₂ described throughout this study, and is in accordance with the crucial role of Kv₁ channels in tuning CA3 pyramidal neurons excitability (Rama et al., 2017) and the implication of dendritic D-type potassium currents (Metz et al., 2007) and Kv₂ channels (Raus Balind et al., 2019) in promoting bursting patterns in hippocampal neurons. Our results are also in agreement with other described forms of increased excitability in *Lgi1*⁻/⁻ mice (Yu et al., 2010) (Zhou et al., 2023) (Boillot et al., 2016), and with the observed increase in excitability upon the treatment of slices with LGI1 antibodies (Lalic et al., 2011) (Kornau et al., 2020) (Extremet et al., 2022). As expected, there was no change in AP waveform since the broadening of the AP due to the loss or pharmacological inhibition of Kv₁.₁ is only noticeable in presynaptic, but not in somatic APs (Vivekananda et al., 2017).

Although these promising results deserve further investigation, it is widely accepted that synchronized bursting is the cellular substrate for interictal spikes, which, in turn, constitute a hallmark of epilepsy (Traub and Wong, 1982) (Xu and Clancy, 2008). Given the high degree of recurrent connectivity in the CA3 hippocampal region (Mitra et al., 2011) (Guzman et al., 2016), it is tempting to propose a cellular landscape in which the abnormally increased burst firing, in combination with a lower firing threshold in *Lgi1*⁻/⁻ CA3 pyramidal neurons, triggers the epileptogenic phenotype observed in *Lgi1*⁻/⁻ mice.

### CRediT authorship contribution statement

**Jorge Ramirez-Franco:** Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization, Visualization, Writing – original draft, Writing – review & editing. **Kévin Debreux:** Data curation, Investigation. **Marion Sangiardi:** Investigation, Methodology. **Maya Belghazi:** Data curation, Investigation, Writing – original draft. **Yujin Kim:** Resources. **Suk-Ho Lee:** Resources. **Christian Lévêque:** Conceptualization, Validation, Writing – original draft, Writing – review & editing. **Michael Seagar:** Conceptualization, Writing – original draft, Writing – review & editing. **Oussama El Far:** Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.

### Declaration of competing interest

The authors declare that they have no conflict of interest.

### Data availability

All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. The proteomic data were deposited on the PRIDE website (https://www.ebi.ac.uk/pride/archive) with the dataset identifier PXD043649.

### Acknowledgements

We thank Stephanie Baulac for sharing the *Lgi1*⁻/⁻ mice strain (Chabrol et al., 2010) and Jeffrey Martens (University of Michigan, USA) for providing Kv1 clones (Jenkins et al., 2011). This work was supported by the Institut National de la Santé et de la Recherche Médicale INSERM), Aix-Marseille Université (AMU) and the Agence Nationale de la Recherche (ANR) (grant ANR-17-CE16-0022). The postdoctoral financial support of J.R.F. was from the ANR (grant ANR-17-CE16-0022). The PhD thesis by K.D. was supported by a fellowship from the French Ministry of Research (MESRI).

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.nbd.2024.106513.

### References

Akhtar, S., et al., 1999. A functional spliced-variant of beta 2 subunit of Kv1 channels in C6 glioma cells and reactive astrocytes from rat lesioned cerebellum. Biochemistry 38, 16984-16992.

Allen, A.S., et al., 2013. De novo mutations in epileptic encephalopathies. Nature 501, 217-221.

Bhadra, P., et al., 2021. Quantitative proteomics and differential protein abundance analysis after depletion of putative mRNA receptors in the ER membrane of human cells identifies novel aspects of mRNA targeting to the ER. Molecules 26.

Bishop, H.I., et al., 2015. Distinct cell- and layer-specific expression patterns and independent regulation of Kv2 channel subtypes in cortical pyramidal neurons. J. Neurosci. 35, 14922-14942.

Boillot, M., et al., 2014. Glutamatergic neuron-targeted loss of LGI1 epilepsy gene results in seizures. Brain : J. Neurol. 137, 2984-2996.

Boillot, M., et al., 2016. LGI1 acts presynaptically to regulate excitatory synaptic transmission during early postnatal development. Sci. Rep. 6, 21769.

Bolte, S., Cordelieres, F.P., 2006. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213-232.

Browne, D.L., et al., 1994. Episodic ataxia/myokymia syndrome is associated with point mutations in the human potassium channel gene, KCNA1. Nat. Genet. 8, 136-140.

Calabrese, B., Halpain, S., 2015. Differential targeting of dynamin-1 and dynamin-3 to nerve terminals during chronic suppression of neuronal activity. Mol. Cell. Neurosci. 68, 36-45.

Chabrol, E., et al., 2010. Electroclinical characterization of epileptic seizures in leucine-rich, glioma-inactivated 1-deficient mice. Brain : J. Neurol. 133, 2749-2762.

Chen, B., et al., 2014. The WAVE regulatory complex links diverse receptors to the actin cytoskeleton. Cell 156, 195-207.

Cho, S.J., et al., 2011. Septin 6 regulates the cytoarchitecture of neurons through localization at dendritic branch points and bases of protrusions. Mol. Cell 32, 89-98.

Clemens Grisham, R., et al., 2013. Mutations in ap1b1 cause mistargeting of the Na(+)/K (+)-ATPase pump in sensory hair cells. PLoS One 8, e06866.

Connors, E.C., et al., 2008. Homeostatic regulation of Kv1.2 potassium channel trafficking by cyclic AMP. J. Biol. Chem. 283, 3445-3453.

Cudmore, R.H., et al., 2010. Spike-time precision and network synchrony are controlled by the homeostatic regulation of the D-type potassium current. J. Neurosci. 30, 12885-12895.

Debanne, D., et al., 2011. Axon physiology. Physiol. Rev. 91, 555-602.

Di Giovanni, J., et al., 2010. V-ATPase membrane sector associates with synaptobrevin to modulate neurotransmitter release. Neuron 67, 268-279.

Ding, J., et al., 2016. Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 535, 111-116.

Doring, J.H., et al., 2021. Refining genotypes and phenotypes in KCNA2-related neurological disorders. Int. J. Mol. Sci. 22.

Du, J., et al., 2000. Frequency-dependent regulation of rat hippocampal somato-dendritic excitability by the K⁺ channel subunit Kv2.1. J. Physiol. 522 Pt 1, 19-31.

Dyhrfjeld-Johnsen, J., et al., 2010. Interictal spikes precede ictal discharges in an organotypic hippocampal slice culture model of epileptogenesis. J. Clin. Neurophysiol.: Off. Publicat. American Electroencephalograph. Soc. 27, 418-424.

Eberhard, D.A., et al., 1994. Alterations of annexin expression in pathological neuronal and glial reactions. Immunohistochemical localization of annexins I, II (p36 and p11 subunits), IV, and VI in the human hippocampus. Am. J. Pathol. 145, 640-649.

Ebrahimi, M., et al., 2016. Astrocyte-expressed FABP7 regulates dendritic morphology and excitatory synaptic function of cortical neurons. Glia 64, 48-62.
not fully rescue the life span of mice (Zhou et al., 2023). Further investigations are needed to address whether LGI1 is directly implicated in modulating Kv₂ expression levels and whether Kv₁ downregulation is parallel to or consequent to Kv₂ downregulation.

In the light of the concomitant decrease in Kv₁ and Kv₂ expression levels, we addressed the intrinsic excitability of CA3 pyramidal neurons of *Lgi1*⁻/⁻. We and others have previously shown that LGI1 is particularly enriched in CA3 hippocampal subregion (Herranz-Perez et al., 2010) (Smedfors et al., 2018) (Ramirez-Franco et al., 2022) and that LGI1 is crucial in controlling neuronal excitability in CA3 pyramidal neurons (Seagar et al., 2017) (Kornau et al., 2020) (Extremet et al., 2022). Moreover, LGI1 antibody-mediated limbic encephalitis is coincident with focal CA3 hippocampal atrophy in human patients (Miller et al., 2017). Although we have previously characterized the intrinsic excitability in pyramidal CA3 neurons of *Lgi1*⁻/⁻ cultured hippocampal slices (Seagar et al., 2017), it has been proposed that cultured hippocampal slices spontaneously develop epileptiform activity (Dyhrfjeld-Johnsen et al., 2010). To eliminate a potential bias introduced by the use of organotypic cultures, and to gain further understanding of the mechanisms underlying LGI1 deficiency-mediated epilepsy, we analysed intrinsic excitability of CA3 pyramidal neurons from acute slices.

We observed an increase in neuronal burst firing propensity and intrinsic excitability defined as a decrease in the rheobase and a shortened latency to the first spike. This gain in excitability converges with the decrease in Kv₁.₁ and Kv₁.₂ as well as Kv₂ described throughout this study, and is in accordance with the crucial role of Kv₁ channels in tuning CA3 pyramidal neurons excitability (Rama et al., 2017) and the implication of dendritic D-type potassium currents (Metz et al., 2007) and Kv₂ channels (Raus Balind et al., 2019) in promoting bursting patterns in hippocampal neurons. Our results are also in agreement with other described forms of increased excitability in *Lgi1*⁻/⁻ mice (Yu et al., 2010) (Zhou et al., 2023) (Boillot et al., 2016), and with the observed increase in excitability upon the treatment of slices with LGI1 antibodies (Lalic et al., 2011) (Kornau et al., 2020) (Extremet et al., 2022). As expected, there was no change in AP waveform since the broadening of the AP due to the loss or pharmacological inhibition of Kv₁.₁ is only noticeable in presynaptic, but not in somatic APs (Vivekananda et al., 2017).

Although these promising results deserve further investigation, it is widely accepted that synchronized bursting is the cellular substrate for interictal spikes, which, in turn, constitute a hallmark of epilepsy (Traub and Wong, 1982) (Xu and Clancy, 2008). Given the high degree of recurrent connectivity in the CA3 hippocampal region (Mitra et al., 2011) (Guzman et al., 2016), it is tempting to propose a cellular landscape in which the abnormally increased burst firing, in combination with a lower firing threshold in *Lgi1*⁻/⁻ CA3 pyramidal neurons, triggers the epileptogenic phenotype observed in *Lgi1*⁻/⁻ mice.

## CRediT authorship contribution statement

**Jorge Ramirez-Franco:** Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization, Visualization, Writing – original draft, Writing – review & editing. **Kévin Debreux:** Data curation, Investigation. **Marion Sangiardi:** Investigation, Methodology. **Maya Belghazi:** Data curation, Investigation, Writing – original draft. **Yujin Kim:** Resources. **Suk-Ho Lee:** Resources. **Christian Lévêque:** Conceptualization, Validation, Writing – original draft, Writing – review & editing. **Michael Seagar:** Conceptualization, Writing – original draft, Writing – review & editing. **Oussama El Far:** Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.

## Declaration of competing interest

The authors declare that they have no conflict of interest.

## Data availability

All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. The proteomic data were deposited on the PRIDE website (https://www.ebi.ac.uk/pride/archive) with the dataset identifier PXD043649.

## Acknowledgements

We thank Stephanie Baulac for sharing the *Lgi1*⁻/⁻ mice strain (Chabrol et al., 2010) and Jeffrey Martens (University of Michigan, USA) for providing Kv1 clones (Jenkins et al., 2011). This work was supported by the Institut National de la Santé et de la Recherche Médicale INSERM), Aix-Marseille Université (AMU) and the Agence Nationale de la Recherche (ANR) (grant ANR-17-CE16-0022). The postdoctoral financial support of J.R.F. was from the ANR (grant ANR-17-CE16-0022). The PhD thesis by K.D. was supported by a fellowship from the French Ministry of Research (MESRI).

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.nbd.2024.106513.

## References

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Allen, A.S., et al., 2013. De novo mutations in epileptic encephalopathies. Nature 501, 217-221.

Bhadra, P., et al., 2021. Quantitative proteomics and differential protein abundance analysis after depletion of putative mRNA receptors in the ER membrane of human cells identifies novel aspects of mRNA targeting to the ER. Molecules 26.

Bishop, H.I., et al., 2015. Distinct cell- and layer-specific expression patterns and independent regulation of Kv2 channel subtypes in cortical pyramidal neurons. J. Neurosci. 35, 14922-14942.

Boillot, M., et al., 2014. Glutamatergic neuron-targeted loss of LGI1 epilepsy gene results in seizures. Brain : J. Neurol. 137, 2984-2996.

Boillot, M., et al., 2016. LGI1 acts presynaptically to regulate excitatory synaptic transmission during early postnatal development. Sci. Rep. 6, 21769.

Bolte, S., Cordelieres, F.P., 2006. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213-232.

Browne, D.L., et al., 1994. Episodic ataxia/myokymia syndrome is associated with point mutations in the human potassium channel gene, KCNA1. Nat. Genet. 8, 136-140.

Calabrese, B., Halpain, S., 2015. Differential targeting of dynamin-1 and dynamin-3 to nerve terminals during chronic suppression of neuronal activity. Mol. Cell. Neurosci. 68, 36-45.

Chabrol, E., et al., 2010. Electroclinical characterization of epileptic seizures in leucine-rich, glioma-inactivated 1-deficient mice. Brain : J. Neurol. 133, 2749-2762.

Chen, B., et al., 2014. The WAVE regulatory complex links diverse receptors to the actin cytoskeleton. Cell 156, 195-207.

Cho, S.J., et al., 2011. Septin 6 regulates the cytoarchitecture of neurons through localization at dendritic branch points and bases of protrusions. Mol. Cell 32, 89-98.

Clemens Grisham, R., et al., 2013. Mutations in ap1b1 cause mistargeting of the Na(+)/K (+)-ATPase pump in sensory hair cells. PLoS One 8, e06866.

Connors, E.C., et al., 2008. Homeostatic regulation of Kv1.2 potassium channel trafficking by cyclic AMP. J. Biol. Chem. 283, 3445-3453.

Cudmore, R.H., et al., 2010. Spike-time precision and network synchrony are controlled by the homeostatic regulation of the D-type potassium current. J. Neurosci. 30, 12885-12895.

Debanne, D., et al., 2011. Axon physiology. Physiol. Rev. 91, 555-602.

Di Giovanni, J., et al., 2010. V-ATPase membrane sector associates with synaptobrevin to modulate neurotransmitter release. Neuron 67, 268-279.

Ding, J., et al., 2016. Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 535, 111-116.

Doring, J.H., et al., 2021. Refining genotypes and phenotypes in KCNA2-related neurological disorders. Int. J. Mol. Sci. 22.

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**Supplementary information**

**The downregulation of Kv₁ channels in *Lgi1*⁻/⁻ mice is accompanied by a profound modification of its Interactome and a parallel decrease in Kv₂ channels**

Jorge Ramirez-Franco<sup>a*</sup>, Kévin Debreux<sup>a</sup>, Marion Sangiardia<sup>a</sup>, Maya Belghazib<sup>b</sup>, Yujin Kim<sup>c</sup>, Suk-Ho Lee<sup>c</sup>, Christian Lévêque<sup>a</sup>, Michael Seagar<sup>a</sup>, Oussama El Fara<sup>a*</sup>

<sup>a</sup>INSERM UMR_S 1072, Unité de Neurobiologie des canaux Ioniques et de la Synapse, Aix-Marseille Université, 13015 Marseille, France

<sup>b</sup>Marseille Protéomique (MaP), Plateforme Protéomique IMM, CNRS FR3479, Aix-Marseille Université, 31 Chemin Joseph Aiguier 13009 Marseille, France

<sup>c</sup>Department of Physiology, Cell Physiology Lab, Seoul National University College of Medicine

103 Daehak-ro, Jongno-gu, Seoul, 03080, South Korea

*Corresponding author <oussama.el-far@inserm.fr>; <jose-jorge.ramirez-franco@univ-amu.fr>

Supplementary Fig. S1; Supplementary Fig. S2; Supplementary Fig. S3; Supplementary Fig. S4; Supplementary Table S1; Supplementary Table S2; Supplementary Table S3; Supplementary sheet; Raw images of WB for Fig. 1; Raw images of WB for Fig. 2; Raw images of WB for Fig. 3; Raw images of WB for Fig. 4; Graphical abstract
**Supplementary information**

**The downregulation of Kv₁ channels in *Lgi1*⁻/⁻ mice is accompanied by a profound modification of its Interactome and a parallel decrease in Kv₂ channels**

Jorge Ramirez-Franco<sup>a*</sup>, Kévin Debreux<sup>a</sup>, Marion Sangiardia<sup>a</sup>, Maya Belghazib<sup>b</sup>, Yujin Kim<sup>c</sup>, Suk-Ho Lee<sup>c</sup>, Christian Lévêque<sup>a</sup>, Michael Seagar<sup>a</sup>, Oussama El Fara<sup>a*</sup>

<sup>a</sup>INSERM UMR_S 1072, Unité de Neurobiologie des canaux Ioniques et de la Synapse, Aix-Marseille Université, 13015 Marseille, France

<sup>b</sup>Marseille Protéomique (MaP), Plateforme Protéomique IMM, CNRS FR3479, Aix-Marseille Université, 31 Chemin Joseph Aiguier 13009 Marseille, France

<sup>c</sup>Department of Physiology, Cell Physiology Lab, Seoul National University College of Medicine

103 Daehak-ro, Jongno-gu, Seoul, 03080, South Korea

*Corresponding author <oussama.el-far@inserm.fr>; <jose-jorge.ramirez-franco@univ-amu.fr>

Supplementary Fig. S1; Supplementary Fig. S2; Supplementary Fig. S3; Supplementary Fig. S4; Supplementary Table S1; Supplementary Table S2; Supplementary Table S3; Supplementary sheet; Raw images of WB for Fig. 1; Raw images of WB for Fig. 2; Raw images of WB for Fig. 3; Raw images of WB for Fig. 4; Graphical abstract
<figure><img src="image_24.png" /><figcaption>**Supplementary Fig. S1.** Antigen retrieval is necessary for specific Kv₁.₂ detection in brain slices. Fixed brains of P14-P16 C57BL/6 wild-type mice were sliced and probed with anti Kv₁.₁ and AnkG antibodies in the absence (upper panels) or presence (lower panels) of antigen retrieval. Note that AnKG staining was not different in both cases while Kv₁.₁ staining was only possible after antigen retrieval. Inset boxes represent the regions depicted in Figure 1C. Scale bar = 12.5 µm</figcaption></figure>
<figure><img src="image_24.png" /><figcaption>**Supplementary Fig. S1.** Antigen retrieval is necessary for specific Kv₁.₂ detection in brain slices. Fixed brains of P14-P16 C57BL/6 wild-type mice were sliced and probed with anti Kv₁.₁ and AnkG antibodies in the absence (upper panels) or presence (lower panels) of antigen retrieval. Note that AnKG staining was not different in both cases while Kv₁.₁ staining was only possible after antigen retrieval. Inset boxes represent the regions depicted in Figure 1C. Scale bar = 12.5 µm</figcaption></figure>
<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /><figcaption>**Supplementary Fig. S2.** empAI ratios histogram of Kv₁ partners in WT and *Lgi1*⁻/⁻. a) Principle up and downregulated Kv₁ partners in WT and *Lgi1*⁻/⁻. b) Histograms of the log of ratios differences allowing to identify at a glance the fold change in partners enrichment. c) Venns diagram of Kv₁ partners in this study.</figcaption></figure>
A

<figure><img src="image_25.png" /></figure>

B

<figure><img src="image_26.png" /></figure>

C

WT = 67 proteins

<figure><img src="image_27.png" /></figure>

KO = 80 proteins

**Supplementary Fig. S2.** empAI ratios histogram of Kv₁ partners in WT and *Lgi1*⁻/-. a) Principle up and downregulated Kv₁ partners in WT and *Lgi1*⁻/-. b) Histograms of the log of ratios differences allowing to identify at a glance the fold change in partners enrichment. c) Venns diagram of Kv₁ partners in this study.
PMCA4 / GAPDH

<figure><img src="image_28.png" /><figcaption>**Supplementary Fig. S3.** PMCA4/AT2B4 expression levels are not modified in *Lgi1*⁻/⁻.</figcaption></figure>

The expression level of PMCA4 was probed by Western blot on brain homogenates of WT and *Lgi1*⁻/⁻. Expression levels normalized to GAPDH signal are represented in histograms WT 2.62 ± 0.17 vs *Lgi1*⁻/⁻ 2.71 ± 0.43 (p = 0.47152; n = 6; Two-tailed Mann-Whitney U test).
PMCA4 / GAPDH

<figure><img src="image_28.png" /><figcaption>**Supplementary Fig. S3.** PMCA4/AT2B4 expression levels are not modified in *Lgi1*⁻/⁻.</figcaption></figure>

The expression level of PMCA4 was probed by Western blot on brain homogenates of WT and *Lgi1*⁻/⁻. Expression levels normalized to GAPDH signal are represented in histograms WT 2.62 ± 0.17 vs *Lgi1*⁻/⁻ 2.71 ± 0.43 (p = 0.47152; n = 6; Two-tailed Mann-Whitney U test).
<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /><figcaption>**Supplementary Fig. S4.** Exclusive Kv₁.₂ partners in WT and *Lgi1*⁻/⁻. Histograms showing exclusive immunoprecipitated protein partners of Kv₁.₂ from hippocampal homogenates in WT (top, green bars) and in *Lgi1*⁻/⁻ (bottom, red bars).</figcaption></figure>
<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /><figcaption>**Supplementary Fig. S4.** Exclusive Kv₁.₂ partners in WT and *Lgi1*⁻/⁻. Histograms showing exclusive immunoprecipitated protein partners of Kv₁.₂ from hippocampal homogenates in WT (top, green bars) and in *Lgi1*⁻/⁻ (bottom, red bars).</figcaption></figure>