A hyperglycosylated form of Kv 1.2 upregulated in LGI1 knockout mice

A hyperglycosylated form of Kv1.2 upregulated in LGI1 knockout mice | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A hyperglycosylated form of Kv 1.2 upregulated in LGI1 knockout mice View ORCID Profile Jorge Ramirez-Franco , Marion Sangiardi , Kévin Debreux , View ORCID Profile Maya Belghazi , View ORCID Profile Christian Lévêque , View ORCID Profile Michael Seagar , View ORCID Profile Oussama El Far doi: https://doi.org/10.1101/2025.10.29.685130 Jorge Ramirez-Franco 1 Aix-Marseille Univ , INSERM U1072, UNIS, Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jorge Ramirez-Franco Marion Sangiardi 1 Aix-Marseille Univ , INSERM U1072, UNIS, Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kévin Debreux 1 Aix-Marseille Univ , INSERM U1072, UNIS, Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Maya Belghazi 3 Marseille Protéomique (MaP), Plateforme Protéomique IMM, CNRS FR3479, Aix-Marseille Université , 31 Chemin Joseph Aiguier 13009 Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Maya Belghazi Christian Lévêque 1 Aix-Marseille Univ , INSERM U1072, UNIS, Marseille, France 2 Aix-Marseille Univ , INSERM U1325, DyNaMo, Turing centre for living systems , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Christian Lévêque Michael Seagar 1 Aix-Marseille Univ , INSERM U1072, UNIS, Marseille, France 2 Aix-Marseille Univ , INSERM U1325, DyNaMo, Turing centre for living systems , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Michael Seagar Oussama El Far 1 Aix-Marseille Univ , INSERM U1072, UNIS, Marseille, France 2 Aix-Marseille Univ , INSERM U1325, DyNaMo, Turing centre for living systems , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Oussama El Far For correspondence: oussama.el-far{at}inserm.fr oussama.el-far{at}univ-amu.fr Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Kv 1 voltage-gated potassium channels determine key functional neuronal properties. Their activity is modulated by subunit composition and post-translational modifications such as phosphorylation and glycosylation. Using an antibody directed against a phosphotyrosine (Y 458 ) located in the C-terminal tail of Kv 1.2 , we identified yet unreported high molecular weight forms of Kv 1.2 among them, a phosphorylated and heavily glycosylated 100 kDa form. Owing to the significant downregulation of Kv 1.2 in LGI1-dependent autosomal dominant lateral temporal lobe epilepsy, we investigated, in total brain and the hippocampal formation of both WT and Lgi1 -/- mice, the distribution of phosphoY 458 Kv 1.2 and we compared their respective proteomic interactomes with those of Kv 1.2 . In addition to major differences between the interactomes of pY 458 Kv 1.2 and Kv 1.2 in WT and Lgi1 -/- , we found a major reshaping of pY 458 Kv 1.2 molecular neighbourhood between WT and Lgi1 -/- as well as a significant upregulation of the glycosylated form in Lgi1 -/- . Introduction Disruption of LGI1 function, whether genetic or autoimmune, results in ADLTE and LGI1-associated limbic encephalitis [ 1 ] [ 2 ]. It is associated with a marked reduction of both total and plasma membrane levels of the delayed rectifying Kv 1 channels [ 1 ] [ 3 ] [ 4 ] [ 5 ]. This decrease is a major contributor to the increased neuronal excitability, and thus presumably to associated epileptic condition. While LGI1 is known to be part of the Kv1-associated proteome [ 6 ] [ 7 ] [ 8 ] [ 9 ], the mechanism by which the loss of this extracellular glycoprotein results in Kv 1 downregulation remains unclear and it can be hypothesised that LGI1 influences the subcellular environment as well as the Kv1 associated-proteome. Ras homolog family member A (RhoA) activity can be linked to receptor tyrosine kinase activity [ 10 ] and has been shown to be highly increased by LGI1 knockout [ 11 ]. LGI1 may also antagonize NgR1-TROY-RhoA signalling pathway, leading to decreased RhoA activation [ 11 ]. The Kv 1 channels are low voltage-activated K + channels involved in the regulation of neuronal excitability by controlling firing patterns and action-potential (AP) thresholds [ 12 ] [ 13 ] [ 14 ]. They are formed by a homomeric or heteromeric co-assembly of four alpha subunits with auxiliary subunits [ 15 ]. D-type α-dendrotoxin-sensitive currents are generated by channels with Kv 1.1 , Kv 1.2 and Kv 1.6 subunits and native Kv 1.2 subunits are present in axonal and synaptic compartments [ 16 ] [ 17 ] as well as at axonal initial segments [ 18 ] [ 1 ]. Mutations in these subunits have been shown to be involved in several neurological disorders [ 19 ] [ 20 ] [ 21 ] [ 22 ] and functional plasticity in neurons involves a dynamic change in the subcellular distribution of potassium channels [ 15 ]. Targeting of Kv 1 to the membrane, as well as subcellular compartments is complex and heterogeneous. Distinct molecular partners are involved in Kv 1 targeting and clustering depending on the neuronal compartment concerned. Phosphorylation of Y 458 in Kv 1.2 plays a negative role in axonal targeting of Kv 1.2 and the Y 458 A mutation was shown to increase axonal targeting [ 17 ]. The phosphorylation of this tyrosine is a target for physiological regulation by GPCR-coupled mechanisms since Y 458 phosphorylation is induced by the muscarinic M1 acetylcholine receptor activation and regulates the interaction of Kv 1.2 with the actin-binding protein cortactin [ 23 ]. Although Kv 1 -associated partners Caspr2 and TAG1 are present at the neuronal axonal initial segment (AIS), these proteins as well as DLG4 [ 24 ] [ 6 ] and ADAM22 [ 6 ] are dispensable for Kv1 recruitment in this compartment [ 16 ] [ 25 ], where other molecular organisers such as DLG2 take over this task [ 24 ] [ 16 ] [ 6 ]. More recently and despite the absence of SCRIB from the proteome of Kv 1.2 [ 26 ], a direct association of Kv 1.2 with SCRIB was suggested to take place in the AIS and together with PSD93 modulate Kv1 clustering [ 27 ]. In addition to the importance of tyrosine phosphorylation in Kv 1.2 clustering, tyrosine kinase-dependent endocytosis of Kv 1.2 has been reported to be a mechanism for Kv 1.2 downregulation [ 28 ]. In order to address the potential importance of Y 458 phosphorylation in determining the molecular environment of Kv 1.2 and therefore gain insight into its importance in the life cycle of Kv 1.2 interactions, we developed a phosphoY 458 Kv 1.2 (pY 458 Kv 1.2 ) antibody and, after immunoprecipitation, compared by mass spectrometry the proteomes of pY 458 Kv 1.2 with the global Kv 1.2 proteome that we have previously described [ 26 ]. Furthermore, since Kv 1.2 is massively downregulated in Lgi1 -/- mice [ 1 ] [ 26 ], we addressed the differences in the pY 458 Kv 1.2 expression and associated proteome between WT and Lgi1 -/- backgrounds. Materials and Methods Antibodies and reagents A rabbit polyclonal antibody (ESM_1a) recognizing specifically the phosphoY 458 (pY 458 ) in the C-terminal peptide (aa 454-SKSD(pY)MEIQEGVNNS-468) of mouse Kv 1.2 was produced. Antibodies recognizing the non-phosphorylated peptide were depleted on an identical but non-phosphorylated peptide column and specific pY 458 antibodies were affinity purified against the pY 458 Kv 1.2 peptide (GeneCust). This antibody was used at 5 µg/ml for western blot analysis and 5 µg were used for immunoprecipitation. Kv 1.1 (K36/15) and Kv 1.2 (K14/16) antibodies were from Neuromab and used at 1 μg/μl for western blots and immunohistofluorescence experiments and 5 μg for immunoprecipitation. Sumo2/3 antibody (4G11E9) was from GenScript biotech and Ubiquitin antibody (sc-166553) from SantaCruz biotechnology, Inc. HRP-coupled secondary antibodies were from Jackson ImmunoResearch. Unless stated otherwise, chemicals were from Sigma-Aldrich, PNGase and calf intestinal phosphase were from New England Biolabs. Dot blots Phosphorylated and non-phosphorylated peptides (5 ng / dot) were dotted onto a nitrocellulose membrane and classical western blot procedure was used to test the recognition of peptides with anti-Kv 1.2 and anti-pY 458 Kv 1.2 peptide antibodies (0.25 µg/ml). Western blots Mouse brains were homogenized in 25 mM Tris-HCl pH 7.4, 150 mM NaCl in the presence of protease and phosphatase inhibitors. Homogenates were recovered in supernatants of a 800×g centrifugation. 40-60 μg of protein were resolved by SDS/PAGE and processed for Western blotting using classical procedures with the indicated antibodies. PNGase treatment After immunoprecipitation, samples were washed and treated with PNGase following the manufacturer instructions. Briefly, solubilized brain extracts from Lgi1 -/- were immunoprecipitated by the pY 458 Kv 1.2 antibody. Immunoprecipitated material was denatured at 100°C for 10 min and divided in multiple identical aliquots. All aliquots were treated exactly the same way in the presence or absence of PNGase (1µl /20 µg of proteins), incubated for 1h at 37°C and analysed by Western blot. Experiments were performed in triplicates. Identification by mass spectrometry of high molecular weight forms of Kv 1.2 Brain homogenates were solubilized in Tris 25 mM pH 7.4, NaCl 150 mM CHAPS 1% in the presence of phosphatase and protease inhibitors. 4 mg were subjected to immunoprecipitation by 40 µg of polyclonal Kv 1.2 antibodies. Immunoprecipitated material was denatured at 55°C for 15 min and resolved by electrophoresis on 8% polyacrylamide gel. The migration was stopped when the molecular weight marker of 55 kDa migrated out of the gel. The gel lane was then sliced into 9 distinct pieces along the migration axis and the presence of Kv 1.2 in each slice was verified by mass spectrometry. Gel bands were processed as previously described [ 26 ]. Briefly, gel slices were reduced with dithiothreitol, alkylated with iodoacetamide and digested overnight at 37° in 25 mM ammonium bicarbonate buffer pH 7.4 using Trypsin/Lys-C mix (Promega, Madison, USA). Peptides were extracted three times using 50% acetonitrile (v/v) in water containing 0.1% (v/v) formic acid and dried in a SpeedVac concentrator. Samples were reconstituted in loading buffer (2% acetonitrile (v/v) in water containing 0.1% (v/v) trifluoroacetic acid) and analyzed by LC-MS/MS (nanoHPLC Neo Vanquish coupled to Qexactive Plus from Thermo Fisher Scientific, San Jose, USA). Peptides were first loaded onto a trap column (PepMap Neo 300 µm x 5mm, C18 5µm, 100Ǻ) then separated on an analytical column (EASY-spray PepMap Neo, 75µm x 500 mm, C18 2µm, 100Ǻ) using the following gradient: from 2% to 25% of mobile phase B (20% water, 80% acetonitrile/0.1% formic acid) in A (0.1% formic acid in water) over 90 min, then to 50% B over 20 min. The mass spectrometer was operated in Data Dependant Acquisition positive mode. Full MS scans were acquired at 70 000 resolution (m/z range 350-1900, Auto Gain Control target 3×106) followed by MSMS scans of the top 10 most intense ions (17 500 resolution, isolation window 2m/z, Auto Gain Control 1×105, normalized collision energy 27). Data were searched against the Mus musculus Uniprot database (TaxID=10090, v2025-02-05) using Proteome Discoverer 3.0 software (Thermo Fisher Scientific, San Jose, USA). The mass spectrometry proteomics data have been deposited to the ProteomeXchange consortium via the PRIDE [ 29 ] partner repository with the data set identifier PXD069032. Biochemical sample preparation, immunoprecipitations and mass spectrometry of pKv 1.2 proteome Sample preparation, immunoprecipitation as well as mass spectrometry were performed as in [ 26 ]. All samples were treated in triplicates. Protein quantification was based on the exponentially modified Protein Abundance Index (emPAI) obtained for each identified protein. Three biological samples from WT and Lgi1 -/- were immunoprecipitated using pY 458 Kv 1.2 antibody. Two parallel biological samples from each genotype were immunoprecipitated using control rabbit antibodies (NIAB). Data were analyzed and for each protein in the partners lists, the emPAI values from the NIAB samples were averaged; if a protein was detected in only one NIAB sample, the missing value was treated as zero before averaging resulting in: Avg emPAI(NIAB) = [ emPAI(NIAB 1) + emPAI(NIAB2) ] / 2 or Avg emPAI(NIAB) = [ emPAI(NIAB present) + 0 ] / 2 For each pY 458 Kv 1.2 antibody sample, the average NIAB emPAI value for a given protein was subtracted to yield a background-corrected emPAI value (corrected emPAI). Subsequently, the ratio between the pY 458 Kv 1.2 antibody and NIAB emPAI values was calculated for each protein: emPAI Ratio = emPAI pY 458 Kv 1.2 / Avg emPAI NIAB . Proteins were retained for further analysis only if they met all of the following criteria: (i) Ratio ≥ 3, (ii) number of unique peptide sequences > 1, (iii) presence in more than one pY 458 Kv 1.2 antibody sample, and (iv) corrected emPAI > 0.1. To remove highly abundant or nonspecific proteins, a semantic filter was applied. Proteins whose descriptions contained any of the following terms were excluded from the analysis: “Complement,” “DNA binding protein”, “Initiation,” “Elongation,” “Haemoglobin,” “Immunoglobulin,” “Keratin”, “Mitochondrial”, “Nuclear,” “RNA,” “RNA splicing”, “exonuclease”, “Ribosomal,” “Transcription,” “Tubulin,” or “Transcriptional.” Proteins that passed both the quantitative and semantic filters were retained, and their corrected emPAI values were averaged across replicates for further analysis. Candidate proteins were classified using the Genecards and pantherdb web sites ( www.genecards.org ; www.pantherdb.org ) and association networks were analysed using the String protein server ( https://string-db.org ). 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). Fixed brains of P14-P16 C57BL/6 wild-type mice or Lgi1 -/- littermates of either sex were sliced and stained as described in [ 9 ]. Three animals per genotype were used for each of the immunohistofluorescence conditions tested. Image analysis For quantification of immunolabeling differences in WT and Lgi1 -/- animals, two different strategies were used. In panel C of Figure 5 , three straight lines were placed along the different CA3 strata in each field of view (n=3 animals, n=6 slices). Fluorescent values were collected as arbitrary fluorescent units (afu) and normalized to the first point of the WT traces. For the panels 5D and 5E, 5 random ROIs (Regions of Interest) where placed in each of the CA3 hippocampal strata (n=3 animals, n=6 Slices). Fluorescence values where collected and normalized to the average fluorescence value of WT in each of the strata. For co-localization analysis, the Just Another Colocalization Plugin (JACOP) was used in its BIOP version [ 30 ] and the Persons’ Correlation Coefficients (PCC) were computed over z-stacks consisting of 10 slices considering z-slices separately but averaging values in a single data point. Results Identification of phosphorylated high molecular weight forms of Kv 1.2 We generated a polyclonal antibody recognizing the phosphorylated form of Kv 1.2 on Y 458 (pY 458 Kv 1.2 ). The specificity of this antibodies was first addressed by dot blot. As shown in ESM_1a, pY 458 Kv 1.2 antibodies recognize the phosphorylated peptide immunogen but not the very same immunogen in its non-phosphorylated state. Of note, our previously reported antibody, generated against the non-phosphorylated peptide [ 26 ], cross-reacts with the phosphorylated peptide in dot blots and therefore recognise both peptides (ESM_1a). Sequence alignment of Kv 1 C-terminal sequences shows a significant similarity in the Y 458 environment between Kv 1.1 , Kv 1.2 , Kv 1.3 and Kv 1.4 , (ESM_1b) and suggests that the pY 458 Kv 1.2 antibody may cross-react with other phosphorylated Kv 1 subunits. Immunoprecipitation experiments followed by mass spectrometry shows that pY 458 Kv 1.2 antibody captures only Kv 1.1 and Kv 1.2 from WT mice and the total absence of other Kv 1 subunits (ESM_1c) that were otherwise associated with Kv 1.2 [ 26 ] namely Kv 1.3 , Kv 1.4 , Kv 1.5 and Kv 1 . 6 subunits. These data argue against a potential cross-reactivity of the pY 458 Kv 1.2 antibodies with other Kv 1 alpha subunits than Kv 1.1 . Western blot analysis of brain homogenates (ESM_2) shows that pY 458 Kv 1.2 antibody identifies, in addition to the circa 70 kDa principal band, 3 bands of higher molecular weight (circa 100, 130 and >200 kDa) that are not recognized in homogenates by the K14/16 Kv 1.2 Neuromab Kv 1.2 antibody as well as our previously characterized polyclonal Kv 1.2 antibody [ 26 ]. In order to confirm the identity of Kv 1.2 reactive species at the observed high molecular weights, Kv 1.2 was immunoprecipitated with the polyclonal Kv 1.2 antibody, that, as shown in ESM_1a, recognizes both phosphorylated and non-phosphorylated peptides, and subjected to polyacrylamide gel electrophoresis. Slices at different migration positions of the migration lane were analysed by mass spectrometry for the presence or absence of Kv 1.2 . As shown in Fig. 1a , Kv 1.2 peptides were found at distinct migration levels corresponding to the observed high molecular weight forms of Kv 1.2 . Since Kv 1.2 is massively decreased in Lgi1 -/- we investigated potential modification in the expression of pY 458 Kv 1.2 in Lgi1 -/- . Similar to the previously described expression decrease in the 70 kDa form of Kv 1.2 in Lgi1 -/- , the a=70 kDa pY 458 Kv 1.2 also decrease by at least two-fold (ESM_2). Two of the newly detected protein bands did not vary (c=130 & d=>200 kDa) and one (b=100 kDa) increased by > 70% in Lgi1 -/- (ESM_2) with b/a ratio of 1.68 ± 1.8 in WT and 3.26 ± 0.66 in Lgi1 -/- . Download figure Open in new tab Fig. 1 Identification and glycosylation profile of the molecular entities immunoprecipitated by pY 458 Kv 1.2 . a Kv 1.2 was immunoprecipitated by the polydonal Kv 1.2 antibody from WT brain extracts and subjected to SDS PAGE. Mass spectrometry analysis was performed on the indicated polyacrylamide gel slices and the presence or absence of Kv 1.2 peptides is reported (in red). The position of the found peptides in the linear Kv 1.2 sequence is indicated in the Kv 1.2 sequence (right side). b, Kv 1.2 was immunoprecipitated by the polydonal Kv 1.2 and the immunoprecipitated material was subjected or not to PNGase treatment. Samples were analysed by Western blot with the pY 458 Kv 1.2 . The circa 70 kDa bands were quantified and their intensity plotted in histograms. p values (paired t-test) are reported under each Western blot (n=3) The large increase in the molecular weight of Kv 1.2 is unlikely due to phosphorylation and may indicate additional post-translational modifications such as ubiquitination, sumoylation or glycosylation. An increase in the apparent molecular weight of Kv 1.3 has been reported upon ubiquitination [ 31 ] and a highly glycosylated form of Kv 1.2 with a large increase in the apparent molecular weight was previously reported in a heterologous system [ 32 ]. None of the Kv 1.2 high molecular weight bands recognized and enriched by the pY 458 Kv 1.2 antibody was recognized by ubiquitin or Sumo2/3 antibodies (ESM_3). However, the 100 kDa band completely disappeared after treatment with PNGaseF while the 70 kDa band increased ( Fig. 1b ). This indicates that the 100 kDa species corresponds to a highly glycosylated form of Kv 1.2 . All these data indicate that the antibody raised against the phosphoY 458 in the C-terminal domain of Kv 1.2 recognizes a phosphorylated form of the well characterized 70 kDa but also other high molecular weight forms with potentially differential post-translational modifications. Distribution of pY 458 Kv 1.2 in WT versus Lgi1 -/- hippocampal regions In order to address the localisation of pY 458 Kv 1.2 and its distribution throughout the brain and the hippocampal formation, we performed immunostainings on whole brain slices of WT animals. The staining pattern of the pY 458 Kv 1.2 antibody revealed an intense labeling of myelinated forebrain and midbrain axonal tracts (ESM_ 4 ). Moreover, a more diffuse yet clearly detectable labeling pattern was observed in several hippocampal regions, particularly within the stratum lucidum (SL) of CA3 (ESM_4 and Figs. 2a, 2b , and 2c ). In order to address the differences in the distribution of pY 458 Kv 1.2 in WT and Lgi1 -/- , we performed a comparative analysis of the staining pattern in WT vs Lgi1 -/- hippocampal slices (WT n=3 animals, Lgi1 -/- n=3 animals). As shown in Fig. 2 ( Figs. 2a, 2b , and 2c ), WT hippocampal slices show weak staining, whereas Lgi1 ⁻/⁻ slices exhibit intense labeling across all analyzed CA3 hippocampal strata (SO: Stratum Oriens; SL: Stratum Lucidum; SR: Stratum Radiatum; Figs. 2c and 6d ; n = 3 animals per genotype). We focused our analysis on CA3 due to its enriched expression of LGI1 and the reported selective reduction of Kv 1 -family channel levels in this region in Lgi1⁻ / ⁻ mice [ 1 ] [ 26 ]. Normalized fluorescence values ( Fig. 2e ) confirmed this observation, with significantly increased signals in Lgi1 ⁻/⁻ compared to WT littermates in all layers of CA3: SO (1.55 ± 0.04 vs. 1.00 ± 0.07), SL (2.17 ± 0.05 vs. 1.00 ± 0.07), and SR (2.04 ± 0.07 vs. 1.00 ± 0.07). In order to ascertain whether this increase in pY 458 Kv 1.2 immunoreactivity was of presynaptic or postsynaptic origin, we performed double immunostaining against pY 458 Kv 1.2 and the presynaptic marker synaptophysin (n=2 animals, Fig. 2f ). Co-localization analyses yielded very low and similar Pearson correlation coefficients between the pY 458 Kv 1.2 signal and synaptophysin in both genotypes (PCC=0.0199 ± 0.0185 in WT and PCC=0.0263 ± 0.0146 in Lgi1 ⁻/⁻ mice), indicating that the hippocampal pY 458 Kv 1.2 signal was mostly of non-presynaptic origin. This large increase in pY 458 Kv 1.2 staining in Lgi1 -/- background may reflect the observed increase in the intensity of the glycosylated 100 kDa form of Kv 1.2 by western blot but potentially also a differential accessibility of the anti pY 458 Kv 1.2 epitope between WT and Lgi1 -/- . Download figure Open in new tab Fig. 2 lmmunofluorescence distribution of pY 458 Kv 1.2 in the hippocampal formation of WT and Lgi1 -/- a Full hippocampal immunostaining with anti-pY 458 Kv 1.2 in WT (top) and Lgi1 -/- (bottom) showing pY 458 Kv 1.2 immunoreactivity (green), DAPI (blue), and merged images. Scale bar = 250 µm. b Detailed views of CA3 hippocampal regions showing immunolabeling against pY 458 Kv 1.2 (green), Synaptophysin (red), DAPI (blue), and merged images. Scale bar = 50 µm. c Average line trace quantification of immunolabeling shown as normalized signal of arbitrary fluorescence units (afu). N = 3 animals, n = 6 slices/animal; 3 lines per slice. Mean ± SEM. d Box plots of randomly placed ROls across CA3 hippocampal strata in WT (black dots and boxes) and Lgi1 -/- (red dots and boxes). N = 3 animals; n = 6 slices/animal; 5 ROls per slice and stratum. (SO WT = 28.66 ± 1.97 vs Lgi1 -/- = 44.40 ± 1.04, p = 3.75 x 10 -44 ; SL WT = 28.46 ± 1.87 vs Lgi1 -/- = 61.79 ± 1.54, p = 1.38 x 10 -44 ; SL WT = 17.60 ± 1.17 vs Lgi1 -/- = 35.96 ± 1.18, p = 1.08 x 10 -15 ). p < 0.01, One-way ANOVA followed by Bonferroni’s test for mean comparisons. e Bar graph comparisons of the normalized values shown in panel C for WT (black bars) and Lgi1 -/- (red bars). Data are Mean± SEM: (SO WT= 1.00 ± 0.07 vs Lgi1 -/- = 1.55 ± 0.04, p = 5.41 x 10 -s ; SL WT= 1.00 ± 0.07 vs Lgi1 -/- = 2.17 ± 0.05, p = 2.66 x 10 -35 ; SR WT= 1.00 ± 0.07 vs Lgi1 -/- = 2.04 ± 0.07, p = 6.55 x 10 -29 ). p < 0.01, One-way ANOVA followed by Bonferroni’s test for mean comparisons. f Detailed views of the stratum lucidum of CA3 in WT (top) and Lgi1 -/- (bottom) showing staining for anti-pY 458 Kv 1.2 (green), anti­ Synaptophysin (red), DAPI (blue), and merged images. Right-most panels show a colocalization map using an Intensity Correlation Analysis (ICA) Lookup Table (LUT). Scale bar = 25 µm. g Pearson’s correlation coefficients (PCC) for co-localization of pY 458 Kv 1.2 and Synaptophysin in WT (black bars) and Lgi1 -/- (red bars). N = 2 animals, n = 4 slices/animal. Data are Mean ± SEM. Two-sample t-test; n.s., non-significant (WT= 0.0199 ± 0.0185 vs Lgi1 -/- = 0.0263 ± 0.0146, p = 0.79). Subunit composition of Kv 1 channels containing pY 458 Kv 1.2 Mass spectrometry analysis of immunoprecipitated samples shows an association between Kv 1.2, Kv 1.1 and Kvβ 2 subunits in WT samples. In contrast, neither Kv 1.1 nor Kvβ 2 were recovered in Lgi1 -/- (ESM_1c). Of note, the recovery yield with anti-pY 458 Kv 1.2 (emPAI = 0.36 in WT; emPAI = 0.14 in Lgi1 -/- ) is much smaller than with anti-Kv 1.2 (emPAI = 2.9 in WT and 2.8 in Lgi1 -/- ) [ 26 ]. This difference cannot be attributed to a limited amount of material upon immunoprecipitation since we used an excess of solubilized WT or Lgi1 -/- brain extracts. This indicates thus that pY 458 Kv 1.2 is only a minor component of the entire Kv 1.2 population. Also, while the association of Kvβ 1 & Kvβ 2 with Kv 1.2 is very consequent (Kvβ emPAI > Kvα emPAI) [ 26 ] (ESM_1), the association of Kvβ 2 with pY 458 Kv 1.2 is clearly lower with Kvα emPAI > Kvβ emPAI). Comparison of Kv 1.2 and pY 458 Kv 1.2 proteomes in WT and Lgi1 -/-- Among the partners that were exclusive to Kv 1.2 or pY 458 Kv 1.2 in WT background compared to Lgi1 -/- , none were found common ( Fig. 3a ). Among those that were exclusive to Kv 1.2 or pY 458 Kv 1.2 in Lgi1 -/- compared to WT, only four showed to be common ( Fig. 3b ). Only seven partners were shared between the common partner lists of Kv 1.2 and pY 458 Kv 1.2 across both genotypes, ( Fig. 3c ). Download figure Open in new tab Fig. 3 Comparative listing of Kv 1.2 versus pY 458 Kv 1.2 partners in WT and Lgi -/- . a In WT. b In Lgit-1 -/- . c common partners in both genotypes Association with cytoskeletal elements We previously showed [ 26 ] that LGI1 modulates Kv 1.2 association with the actin network since, in the absence of LGI1, Kv 1.2 loses interaction with cytoskeletal actin but remains associated with adducin and doublecortin ( Fig. 3a, b ). The present data show that in WT, pY 458 Kv 1.2 is not associated with cytoskeletal actin but with actin related protein 1a (actr1a) that is a component of the dynactin complex involved in microtubule based intracellular transport. It is also associated with the spectrin / tau / microtubule network [ 33 ]. The presence of actr1a, spectrin as well as cofilin and other actin regulating proteins in association with pY 458 Kv 1.2 ( Fig. 3a ; Fig. 4 ) in WT but not in Lgi1 -/- may indicate a link between pY 458 Kv 1.2 and endocytic pathways [ 34 ] and highlights an important difference in the fate of Kv 1.2 relative to pY 458 Kv 1.2 as well as pY 458 Kv 1.2 in WT and Lgi1 -/- . Interestingly, the association of pY 458 Kv 1.2 with the microtubule associated tether protein VAPA [ 35 ] and the non-vesicular lipid transfer protein ASTRB [ 36 ] [ 37 ] is strongly decreased in Lgi1 -/- , pointing towards a mislocalization of pY 458 Kv 1.2 in Lgi1 -/- . Download figure Open in new tab Fig. 4 Comparative listing of cytoskeletal elements and endocytotic effectors associated with pY 458 Kv 1.2 in WT and Lgi1 -/- a, b report cytoskeletal links and Clathrin-mediated versus Clathrin-independent endocytotic effectors respectively Comparison of pY 458 Kv 1.2 proteome between WT and Lgi1 -/- shows that, pY 458 Kv 1.2 in WT is associated with indicators of the clathrin-dependent endocytic pathway ( Fig. 4a ), while pY 458 Kv 1.2 in Lgi1 -/- seems to be handled differently ( Fig. 4a ). Upon comparison of all Kv 1.2 and pY 458 Kv 1.2 partners, 15 and 16 partners were common to WT and Lgi1 -/- respectively (ESM_5). Among these, 7 were common in both WT and Lgi1 -/- . Analysis of the Go terms of non-common partners show a particular enrichment of the Go term “cytoskeleton” for pY 458 Kv 1.2 partners in Lgi1 -/- (ESM_5) suggesting that despite the absence of interaction with the actin and spectrin, pY 458 Kv 1.2 in Lgi1 -/- may be subject to a differential regulation of interaction with cytoskeletal links. A closer look at exclusive pY 458 Kv 1.2 partners in Lgi1 -/- ( Fig. 4b ) shows the association with clathrin-independent endocytosis adaptors and cytoskeletal stabilization mediators (Arp3, Actr1a, Rac1 & 3, Add1, Gda, Gas7 and Otub1) suggesting a particular compensatory mechanism stabilizing cytoskeletal anchoring. Arp3 is the ATP-binding component of Arp2/3 complex involved in actin polymerization [ 38 ]: Actr1a / alpha centractin is a component of a multi-subunit complex involved in microtubule based vesicle motility [ 39 ]. Rac1 & 3 are important effectors in actin filaments polymerisation [ 40 ]. Add1 is a membrane-cytoskeleton-associated protein promoting the assembly of the spectrin-actin network [ 41 ] and Gas7 is an actin-filament binding protein [ 42 ]. By its deubiquitination activity, Otub1 modulate the endocytotic fate of certain proteins. Of note, Kv 1.2 and pY 458 Kv 1.2 are associated, only in WT, with Cyfip2 that negatively regulates Rac1-driven cytoskeletal remodelling and the actin-binding protein GAS7 highlighting the importance of LGI1 in the association of Kv 1.2 and pY 458 Kv 1.2 with the cytoskeletal network. Association with septins As previously described [ 26 ], the association of Kv 1.2 with septins is different between WT and Lgi1 -/- . In WT, Kv 1.2 associates with only two septins (8 and 11) while in Lgi1 -/- , three additional septins, the neuron-specific septin 3 implicated in actin filament and microtubule dynamics [ 43 ] as well as septins 6 and 7 are also present ( Fig. 5a ). We find that pY 458 Kv 1.2 immunoprecipitated from WT background is also associated with septins 8 and 11 like Kv 1.2 but also with an additional septin (septin 5) ( Fig. 5a ). Suprisingly, in Lgi1 -/- , pY 458 Kv 1.2 totally loses association with all septins while its non-phosphorylated equivalent is strongly associated with septins 3, 6, 7, 8 and 11. These data suggest that septins may play a crucial role in shaping the localisation of pY 458 Kv 1.2 in Lgi1 -/- , especially through an increase in association with Kv 1.2 and a complete loss of association with pY 458 Kv 1.2 . These data highlight again an important difference in the fate of pY 458 Kv 1.2 between WT and Lgi1 -/- . Download figure Open in new tab Fig. 5 Septins and adaptor proteins associated with Kv 1.2 and pY 458 Kv 1.2 in WT and Lgi1 -/- a Comparative listing of Kv 1.2 and pY 458 Kv 1.2 association with septins b Comparative listing of Kv 1.2 and pY 458 Kv 1.2 association with adaptor proteins Association with adaptor protein Following LGI1 depletion, Kv 1.2 loses association with AP1 and AP3 subunits but still associates with AP2 via AP2a2 [ 26 ] ( Fig. 5b ). We find that AP1, AP2 and AP3 subunits [ 44 ] (AP3b2; neuron-specific subunit of the AP3 complex, AP3d1, AP1b1, AP2b1, AP2a1, AP2m1) are associated with pY 458 Kv 1.2 in both genotypes with no fundamental difference between WT and Lgi1 -/- ( Fig. 5b ). Proteins implicated in LGI1-deficiency associated-pathology We previously described 42 common molecular partners of Kv 1.2 in both WT and Lgi1 -/- genetic backgrounds [ 26 ] (ESM_6). Interestingly, 35 of these partners are not present in the proteome of pY 458 Kv 1.2 in particular ADAM22, DLG1, 3,4 as well as 14-3-3 γ and ε, all implicated in LGI1-deficiency associated-pathology. Association with exo-endocytotic proteins We previously reported that Kv 1.2 is only weakly associated with exo-endocytic markers in WT background and that the absence of Lgi1 -/- induces stronger association with these markers and with the somatodendritic endocytic marker dynamin 3 [ 26 ]. The current analysis shows that the association of Kv 1.2 and pY 458 Kv 1.2 with exo-endocytic markers is different already in WT background with pY 458 Kv 1.2 being significantly associated with exo-endocytic markers ( Fig. 6a ). The association of pY 458 Kv 1.2 with dynamin 1 and not dynamin 3 indicate a fundamental difference in the localisation of the major analysed pool of pY 458 Kv 1.2 in WT suggesting that it is actively endocytosed in presynaptic terminals. pY 458 Kv 1.2 is also associated with the vacuolar protein sorting ortholog 35 (Vps35) that participates in endosomal sorting. Surprisingly, and despite the enrichment of adaptor proteins with pY 458 Kv 1.2 in Lgi1 -/- , dynamin1 association is lost indicating potentially either that the endocytic process is hindered or that it takes place in a dynamin-independent pathway. While the ubiquitin removal enzyme OTUB1 which opposes ubiquitination and prevent degradation, is exclusively associated with pY 458 Kv 1.2 in WT background, the ubiquitin activation E1 enzyme UBA1 is only immunoprecipitated with pY 458 Kv 1.2 in Lgi1 -/- . In parallel to UBA1, the valosin-containing protein (Vcp) is only found in the proteome of pY 458 Kv 1.2 in Lgi1 -/- . Vcp interacts with ubiquitinated proteins helping their extraction from membranes or protein complexes before degradation [ 45 ]. Together, these observations suggest that in Lgi1 -/- , the retrieval of pY 458 Kv 1.2 from membranes becomes dynamin independent. The difference in the association with dynamin may also reflect a change in dynamic regulation (exo/endocytosis) of the glycosylated Kv 1.2 that could account for the plasticity of intrinsic excitability. Download figure Open in new tab Fig. 6 Exo-endocytotic and Rho-cycle-associated proteins associated with pY 458 Kv 1.2 in WT and Lgi1 -/- a Comparative listing of Kv 1.2 and pY 458 Kv 1.2 association with exo-endocytotic proteins, b Comparative listing of pY 458 Kv 1.2 association with Rho-cycle-associated proteins in WT and Lgi1 -/- Rho GTPase-associated pathways with pY 458 Kv 1.2 in Lgi1 -/- Guanine nucleotide exchange factors (GEFs) turn on membrane localized small G-proteins signalling by catalysing the exchange of GDP with GTP, GTPase-activating proteins (GAPs) terminate their signalling by inducing GTP hydrolysis [ 46 ] [ 47 ]. Arhgef2 and Arhgap26 are RhoA effectors implicated in microtubule cytoskeletal dynamics. Upon dissociation from microtubules, Arhgef2 stimulates membrane-bound RhoA leading to actin stress fibre formation that can influence membrane trafficking and endocytosis while Arhgap26 inhibits membrane-bound RhoA activity and stabilises synapses. While we did not observe an association of GEFs and GAPs with Kv 1.2 [ 26 ], the association of pY 458 Kv 1.2 with the RhoA GAP (Arhgap26) occurs in both WT and Lgi1 -/- genotypes, its association with RhoA GEF (Arhgef2) occurs only in WT background ( Fig. 6b ). This association again points towards distinct signalling pathways associated with Kv 1.2 and pY 458 Kv 1.2 and that LGI1 ablation influences the association of pY 458 Kv 1.2 with the Rho GEF Arhgef2. This is especially interesting knowing that RhoA activity is significantly increased in Lgi1 -/- [ 11 ]. The association of Rac1 and Rac3 with pY 458 Kv 1.2 occurs only in Lgi1 -/- and with high emPAIs. Rac 3 has been shown to be important in neuronal spine formation [ 48 ] and its enrichment in pY 458 Kv 1.2 in Lgi1 -/- can be related to the decrease in spine pruning observed in in these animals. While Rac 1 is ubiquitous, Rac3 is expressed in neurons and is strongly expressed in CA1-CA3 in the hippocampus [ 48 ]. Also, Rac1 and microtubules, are major players in clathrin-independent endocytosis [ 49 ]. The enrichment of pY 458 Kv 1.2 proteome with microtubules and Rac1 and the association of pY 458 Kv 1.2 with Rac1 only in Lgi1 -/- again suggest that pY 458 Kv 1.2 has a different fate between WT and Lgi1 -/- and that in the absence of LGI1, it may be endocytosed through clathrin-independent pathways ( Fig. 4b ). RACK1 association with Kv 1.2 and pY 458 Kv 1.2 In contrast to Kv 1.2 and independently from the presence or absence of LGI1, pY 458 Kv 1.2 is associated with a number of kinases and kinase scaffolds ( Fig. 3 , ESM_7) suggesting that it is associated with an enzymatically active environment and kinase hotspots. The WD-repeat family member RACK1 (Receptor for activated C Kinase 1) [ 50 ] is a shuttling and scaffolding protein. It contributes to several aspects of cellular function and is a key mediator of various pathways. It binds and stabilizes active or inactive conformations of its partners (i.e. stabilizing inactive Src and Fyn kinases and active PKCβII). Interestingly, RACK1 is the only common partner between Kv 1.2 in Lgi1 -/- and pY 458 Kv 1.2 in WT. In the list of Kv 1.2 partners specifically acquired in Lgi1 -/- , we found the cAMP-dependent protein kinase catalytic subunit beta (Prkacb) ( Fig. 3b ). This association is compatible with the presence of RACK1 among the list of partners and the known involvement of RACK1 in the adenylate cyclase signalling pathway. In contrast, this protein was not a pY 458 Kv 1.2 partner in WT. Instead, we found a large number of kinases and phosphatases as well as scaffolds and G protein subunits ( Fig. 1c ) compatible the hub function of RACK1. Among these proteins, the Gα (Gnao) and Gi (Gnb2), Gβ (Gnb5), serine/threonine-protein kinase DCLK1, mitogen-activated protein kinase 3, cyclin-dependent-like kinase 5 Cdk5), serine/threonine-protein phosphatase 2A, serine/threonine-protein phosphatase 2B catalytic subunit alpha, the casein kinase II subunit beta (Csnk2b), Wiskott-Aldrich syndrome protein family member 1 (Wasf1), which is a downstream effector of tyrosine kinase receptors and small GTPases implicated in actin cytoskeleton organization. These data highlight a fundamental difference in the signalling platforms associated with Kv 1.2 and pY 458 Kv 1.2 in WT animals. In addition, since Kv 1.2 joins RACK1 platforms in Lgi1 -/- and pY 458 Kv 1.2 leaves them, LGI1 appears to be a key factor in organizing signalling platfoms associated with Kv 1.2 and pY 458 Kv 1.2 channels. CNPase is specifically associated with pY 458 Kv 1.2 in Lgi1 -/- Among the proteins that are specific to pY 458 Kv 1.2 versus Kv 1.2 in Lgi1 -/- , we found 2’,3’-cyclic-nucleotide 3’-phosphodiesterase (CNPase/Cnp or CN37) ( Fig. 3b ). Cnp is the most abundant protein in myelin [ 51 ] and is a potential auto-antigen in multiple sclerosis [ 52 ]. Variability in its expression levels has been linked to major depressive disorder [ 53 ] [ 54 ] and schizophrenia [ 55 ] [ 56 ]. Association of pY 458 Kv 1.2 with microtubule-associated proteins A specific characteristic of pY 458 Kv 1.2 is its association with microtubule-associated proteins ( Fig. 3c ). MAP1b, MAP1 light chain 3a, MAP1a, MAP6, and MAP4 (respectively from the most to the least abundant) were exclusively immunoprecipitated with pY 458 Kv 1.2 from both WT and Lgi1 -/- however, pY 458 Kv 1.2 from Lgi1 -/- was significantly less associated with MAPs (ESM_7). The glycogen synthase kinase-3 beta (GSK3b) that controls the activity of MAP1a and b [ 57 ] [ 58 ] was also recovered although equally enriched from WT and Lgi1 -/- backgrounds. These data suggest that, in Lgi1 -/- , pY 458 Kv 1.2 may be associated with phosphorylated MAP1b that renders microtubules prone to depolymerisation, leading to the recovery of circa 3x less MAP1b than in WT (ESM_7). Upon comparison of Kv 1.2 and pY 458 Kv 1.2 partners in an Lgi1 -/- background ( Fig. 3b ), only four common partners were present: CCT4 (a chaperone T-complex subunit involved in actin and tubulin folding), glyceraldehyde-3-phosphate dehydrogenase (Gapdh/G3P) that can modulate the organization and assembly of the cytoskeleton, the glycolytic enzyme L-lactate dehydrogenase B chain (LDBH) indicator of an oxidative stress and the filamentous actin cross-linking protein myristoylated alanine-rich C-kinase substrate (MARCKS), which is the most prominent cellular substrate for protein kinase C. This comparison again suggests that Kv 1.2 downregulation in Lgi1 -/- is related to perturbation of association with the actin cytoskeleton. The presence of CCT4 at equivalent emPAI levels for phosphorylated and non-phospholytated Kv 1.2 suggests that the absence of LGI1 have similar destabilizing effects on both channel types increasing their clearance [ 59 ]. Partners of pY 458 Kv 1.2 not associated with Kv 1.2 pY 458 Kv 1.2 is associated with several new partners (ESM_7, ESM_8 and ESM_9) that were not present in the Kv 1.2 proteome [ 26 ]. Analysis of these partners in GO terms database shows a diverse molecular interactions and subcellular localisation (ESM_10) suggesting that despite a considerable difference in their proteomes, Kv 1 channels containing pY 458 Kv 1.2 are as widely distributed throughout neuronal compartments as the non-pY 458 Kv 1.2 . Discussion In this study, an affinity purified specific Kv 1.2 pY 458 antibody allowed us to identify minor high molecular weight Kv 1.2 entities that were not detected previously. Among them a hyperglycosylated and tyrosine-phosphorylated (pY 458 ) form of 100 kDa which is upregulated in Lgi1 -/- . We did not detect any posttranslational modification in the other Kv 1.2 entities that could explain their electrophoretic profile and therefore, their apparent high molecular weight may be attributed to aggregation despite the denaturation being performed at relatively low temperature. Mass spectrometry analysis of protein complexes immunoprecipitated by pY 458 Kv 1.2 antibodies from both wild type and Lgi1 -/- backgrounds shows a significantly different profile from those previously described for Kv 1.2 [ 26 ] with antibodies recognizing the same but non-phosphorylated Kv 1.2 peptide (ESM_1). In addition, since the proteome of pY 458 Kv 1.2 in Lgi1 -/- background is different from that of Kv 1.2 in Lgi1 -/- , the phosphorylation of Y 458 may not be the key parameter in Kv 1.2 downregulation in Lgi1 -/- and whether phosphorylation of Kv 1.2 Y 458 is linked to glycosylation cannot be inferred from the current study. Previous work showed that Kv1 glycosylation affects Kv 1 gating properties [ 60 ] and facilitates channel trafficking to the cell membrane and its stabilization [ 61 ]. While a decrease in Kv 1.2 expression level can be directly linked to the reduced D-type current and the increase in neuronal excitability in Lgi1 -/- , the increase in the intensity of the described highly glycosylated form of Kv 1.2 in Lgi1 -/- questions the functional role of these subunits and their participation in the potassium conductance. Recently, we uncovered the changes in expression and distribution of Kv 1.2 in Lgi1 -/- mice compared to their WT littermates [ 26 ]. However, a direct comparison between the staining patterns obtained with the monoclonal Kv 1.2 and the polyclonal pY 458 Kv 1.2 antibodies must be interpreted with caution. Moreover, citrate-buffer heat-induced antigen retrieval was performed for Kv 1.2 but could not be applied to pKv 1.2 , as the phospho-epitope is labile and presumably dephosphorylates under such conditions. Nevertheless, although the overall staining distribution obtained with both antibodies was comparable throughout the hippocampal formation, three main differences were observed between the anti-Kv 1.2 and the anti-pY 458 Kv 1.2 staining. First, anti-Kv 1.2 selectively labels axonal initial segments (AIS) of both, pyramidal cells and parvalbumin interneurons in the hippocampal formation, whereas such a staining pattern was not detected with the pY 458 Kv 1.2 antibody. Second, Kv 1.2 immunostaining was homogeneous through CA3 hippocampal region but markedly heterogeneous in CA1 and the Dentate Gyrus (DG), where it labelled preferentially the Stratum Lacunosum Moleculare (SLM) and the Molecular Layer (ML), respectively. In contrast, pY 458 -Kv 1.2 was rather faint in the hippocampal formation, but showed preferential association with the stratum lucidum (SL) of CA3 and, to a lesser extent, the ML of the DG (see ESM_4). Tyrosine phosphorylation followed by internalization of Kv 1.2 channels has been proposed as a mechanism mediating LTP of intrinsic excitability in CA3 pyramidal neurons [ 62 ] [ 63 ]. As such, the increased immunoreactivity in the SL of CA3, which is at least partially from a non-presynaptic origin (see Fig. 2 ), could underlie this phenomenon. Lastly, the pY 458 -Kv 1.2 , shows a prominent association with myelinated fiber bundles (ESM_4), a feature absents in Kv 1.2 immunostainings. We and others have shown that the detection of Kv 1 channels by immunofluorescence is strongly influenced by Kv 1 subcellular distribution and the molecular environment of the epitopes [ 64 ] [ 26 ]. Thus, a modified molecular environment of pY 458 Kv 1.2 could partially account for the reduced accessibility of the antibody in WT compared to Lgi1 -/- . While the staining of Kv 1.2 and pY 458 -Kv 1.2 can provide complementary information, they cannot be quantitatively compared in a straightforward manner. Any observed differences in staining intensity or distribution should therefore be interpreted as reflecting differences in epitope availability rather than absolute changes in protein quantity. In contrast to the previously described Kv 1 . 2 channels subunit composition where several Kvα are part of the channel and the Kvβ 2 /Kvα 1.2 ratio is >4 in WT animals [ 26 ], pY 458 Kv 1.2 associated subunit composition is rather limited to Kv 1 . 1 and Kvβ 2 in WT and Kvβ 2 does not associate with Kv 1 . 2 in Lgi1 -/- . In addition, Kvβ 2 /Kvα 1.2 ratio is around 0.4 (ESM_1). The reason for the limited association of pY 458 Kv 1.2 with other Kv 1 subunits that are present in dendrotoxin sensitive channels is not clear. However, it may be speculated that this might be due to i) conformational changes induced by Y 458 phosphorylation ii) the recruitment of new partners iii) changes in cytoskeletal links driving this modified subunit away from its partner subunits. DLG2 and DLG4 were shown to associate with Kv 1.2 [ 26 ] and PDZ interactions are crucial for Kv 1 surface expression [ 17 ]. However, these interactions are dispensable for Kv 1.2 juxtaparanodal clustering [ 65 ]. More recently a direct association of Kv 1.2 with SCRIB was suggested to take place in the AIS and with PSD93 modulate AIS Kv1 clustering [ 27 ]. In central and peripheral myelinated neurons, the neuronal cell adhesion molecule Caspr2 and the neuro-glial glycosyl-phosphatidyl-inositol-anchored cell adhesion molecule TAG1 play an important role in juxtaparanodal clustering of Kv 1.2 [ 66 ] [ 67 ] and homophilic cis and trans interactions of TAG1 are crucial in this mechanism [ 68 ], controlling thus the internodal resting potential [ 69 ] [ 66 ]. In this context, it was suggested that TAG1-mediated Kv 1.2 clustering could be initiated by the enrichment of TAG1 in lipid rafts, followed by cis and trans homophilic TAG1 interactions and that this clustering is maintained by the local underlying actin cytoskeleton [ 68 ]. The phosphorylation of the C-terminal tyrosine residue Y 458 in Kv 1.2 is required for TAG1-mediated clustering mechanism [ 68 ] but reduces axonal targeting [ 17 ]. Therefore, one would expect pY 458 Kv 1.2 to be less associated with axonal markers. Surprisingly, neither SCRIB nor Caspr2 or TAG1 are found in the list of pY 458 Kv 1.2 partners and the newly identified channel population is not associated with any of the DLG proteins and is associated in WT with Kif5 and Kvβ 2 suggesting their presence in an axonal pool [ 70 ] [ 71 ]. Altogether, the current data set suggests that the newly identified Kv 1.2 population has fundamentally different molecular partnerships compared to the previously described one. Immunofluorescence data corroborate the mass spectrometry findings and show that the hyperglycosylated pY 458 Kv 1.2 occurs in axonal compartments. This localisation may be influenced by the hetero-multimeric nature of Kv 1 channel subunits and their phosphorylation status. Kvβ is important in Kv 1.2 trafficking [ 72 ] and the functional interaction of Kvα and Kvβ subunits depends on their respective phosphorylation status [ 73 ]. A continuum of S/T as well as Y residues phosphorylation may co-exist and therefore co-influence membrane expression, compartment targeting as well as binding to molecular partners. Independently of Kvβ catalytic keto-reductase activity, its NADP + binding property is important in Kv 1.2 trafficking [ 72 ]. Only in Lgi1 -/- , pY 458 Kv 1.2 is associated with thioredoxin (emPAI 0.68) and aldolase (emPAI 0.43) (ESM_9), two metabolic enzymes linked to NADP. Interestingly, it was shown that aldolase may be inactivated by oxidative conditions and NADPH is able to reactivate it through the NADP-dependent thioredoxin system [ 74 ]. Together, the important recruitment of aldolase and thioredoxin to the proteome of pY 458 Kv 1.2 may highlight a modification of oxidative stress in Lgi1 -/- and suggest the existence of a specific metabolic pathway for pY 458 Kv 1.2 in Lgi1 -/- . It is interesting to note that adducin that mediates actin-spectrin interaction [ 75 ] is found associated with pY 458 Kv 1.2 only in Lgi1 -/- . The phosphorylation of adducin disrupts adducin-mediated actin-spectrin interaction and leads to cytoskeletal reorganisation [ 76 ]. Knowing that actin territories control protrusions, while spectrin ones concentrate in retractile zones [ 77 ], this molecular interaction may be implicated in the cytoskeletal reorganisation in Lgi1 -/- and the decrease in the number of dendritic spines. Septins are GTP-binding cytoskeletal components that assemble to form hetero-oligomeric complexes, filaments, bundles and rings [ 78 ] [ 79 ]. They modulate the functions of actin and microtubules and act as scaffolds [ 79 ] for protein recruitment at the plasma membrane as well as in the cytosol. They bind to phosphoinositides and were shown to form diffusion barriers in dendritic spines [ 79 ] [ 80 ]. They also stabilize and maintain ankyrin G in the axonal initial segments [ 81 ]. In contrast to the association of Kv 1.2 with septins in WT and Lgi1 -/- as well as pY 458 Kv 1.2 in WT, our current analysis shows the total loss of association pY 458 Kv 1.2 with septins in Lgi1 -/- . These data suggest that septins may play a crucial role in shaping the localisation of pY 458 Kv 1.2 . Among the specific partners of pY 458 Kv 1.2 that were absent from the Kv 1.2 proteome, we found 2 proteins associated with the Rho GTPase effectors Arhgef2 and Arhgap26 in WT. In Lgi1 -/- , the association with Arhgef2 is lost with a concomitant and strong association with 2 Rho proteins Rac1 and Rac3 [ 82 ] which is in agreement with the previously described significant increase in RhoA activity in Lgi1 -/- . The Rho family members are molecular switches and their GTPase activity is crucial for an important number of cellular mechanisms from signal transduction to cytoskeleton remodelling [ 82 ] [ 83 ] [ 84 ]. The interaction of LGI1 with NgR1 reduces RhoA signalling promoting synapse formation and stabilization [ 11 ]. Also, RhoA plays a critical role in regulating the membrane flow through the endosomal system modulating the stability of the myelin proteolipid protein at oligodendroglial-membranes in contact with neurons [ 84 ]. Interestingly, GTP-bound RhoA was shown to co-imunoprecipitate with Kv 1.2 and to regulate its activity since it plays a role in GPCR and tyrosine kinase-mediated downregulation of Kv 1.2 activity [ 85 ]. The enzyme SIRT2 (NAD-dependent protein deacetylase sirtuin-2) is functionally associated with the tubulin cytoskeletal reorganisation through its implication in protein deacetylation [ 86 ]. The presence of SIRT2 in the proteome of only WT pY 458 Kv 1.2 (ESM_8) may indicate an active deacetylation process that could affect the pY 458 Kv 1.2 partner Tau ( Fig. 1 ). Therefore, deacetylated Tau may have a reduced capacity to stabilize microtubules [ 87 ] leading to domains with impaired cytoskeletal stability where pY 458 Kv 1.2 is enriched. Some common markers for the regulation of cytoskeletal dynamics and endocytic pathways were shared between Kv 1.2 and pY 458 Kv 1.2 . While Cyfip2 was found specifically associated with Kv 1.2 in WT (emPAI 0.11), it is a partner of pY 458 Kv 1.2 in both WT and Lgi1 -/- genotypes with comparable emPAI values (ESM_7) and this despite that the emPAI value of Kv 1.2 immunoprecipitated by anti-pY 458 Kv 1.2 is 10 times less than that immunoprecipitated by anti-Kv 1.2 [ 26 ]. This may suggest that Cyfip2 is present in immunoprecipitated complexes through an interaction with a common partner to Kv 1.2 and pY 458 Kv 1.2 namely either Gsk3b or Ap1b1 ( Fig. 3a, c and ESM_5). While Ap1b1 is an adaptor protein for endocytosis, Gsk3b is involved in cytoskeletal dynamics [ 88 ] and Cyfip2 is part of the WAVE1 complex and is involved in cytoskeletal dynamics and endocytic trafficking. AP2a2, and AP2b1, implicated in clathrin-mediated endocytosis, were equally enriched in both genotypes. AP3b2 and AP3d1 participate in polarized sorting [ 44 ] and their association with pY 458 Kv 1.2 may indicate a common specific sorting pathway of the phosphorylated Kv 1.2 subunit in both WT and Lgi1 -/- . AP2m1 is responsible for cargo selection and directly interacts with ATP6V1H subunit and the autophagosome marker Map1lc3a [ 89 ] that are both present in the pY 458 Kv 1.2 proteome from both genotypes. This may suggest that autophagosomes are part of the dynamic regulation of pY 458 Kv 1.2 expression Annexin A2, copine 6 and 7 are calcium and lipid binding proteins involved in intracellular signal transduction [ 90 ] [ 91 ]. Also, annexin A2 is involved in endosomal repair following organelle destabilization [ 92 ] and copine 6 and 7 are SNARE binding proteins that monitor spontaneous neurotransmission [ 93 ] [ 94 ]. In Lgi1 -/- , Kv 1.2 is associated with copine 6 and annexin A2 [ 26 ] while pY 458 Kv 1.2 only associates with the brain enriched copine 7. These data highlight again the different fate of pY 458 Kv 1.2 between WT and Lgi1 -/- . We previously showed that Kv 1.2 loses association with myelin associated proteins in Lgi1 -/- [ 26 ] and suggested that one consequence of LGI1 depletion could be the exclusion of Kv 1.2 from myelin containing regions. The association of pY 458 Kv 1.2 with contactin 1, in both genotypes and PDIA3 and SIRT2 in WT may be an indicator of the presence of the newly identified form of Kv 1.2 in myelinated axons [ 95 ]. However, the exclusive association of pY 458 Kv 1.2 in Lgi1 -/- with the myelin protein Cnp [ 96 ] as well as with SERA and ALDOA, that are present in the CNS myelin proteome [ 86 ], may participate stabilizing pY 458 Kv 1.2 in the membrane of Lgi1 -/- . As for Kv 1.2, in Lgi1 -/- the interactome of pY 458 Kv 1.2 shows a major reshaping of its molecular neighbourhood. The major changes in molecules implicated in signalling cascade, the modifications in the association with proteins involved in trafficking and recycling dynamics, the increased links with metabolic/stress-related molecules argue that LGI1 behaves as a local organizer and its deletion results in the rewiring of intracellular Kv 1.2 interactomes. The change in the association with the actin cytoskeleton in Lgi1 -/- suggests a cytoskeletal re-anchoring /stabilization of Kv 1.2 and pY 458 Kv 1.2 . In conclusion, Since glycosylation facilitates Kv 1 activation with lower depolarization intensities and stabilizes the channels in membranes [ 60 ] [ 61 ], the upregulated hyperglycosylated pY 458 Kv 1.2 expression may be a homeostatic attempt from neurons to stabilize membrane Kv 1.2 in response to LGI1 deletion. Of note, protein tyrosine phosphatase receptor type D (PTPRD) was found in a Genome-Wide Association study with LGI1-antibody encephalitis [ 97 ]. Therefore, one may speculate that inhibition of phospho-tyrosine kinase activity could be a way of stabilizing membrane Kv 1.2 avoiding thus massive neuronal excitability perturbations. Future investigations should consider whether this could be a common attempt from biological systems to alleviate excitability increase due to Kv 1.2 expression decrease or blockade. Statements & declarations Author contributions O.EF conceived the study, supervised the entire project, performed the experimental design, data analysis, classification, interpretation and manuscript preparation. M.Se. and C.L. contributed to the design of the study. J.R-F performed mass spectrometry data analysis, immunofluorescence experiments on brain slices, analysed images and prepared immunofluorescence figures. K.D. performed immunoprecipitations. J.R-F and K.D. took care of mice breeding and availability of Lgi1 -/- animals. M. B. performed mass spectrometry experiments. O.EF and M.S. performed biochemical experiments. O.EF wrote the original draft of the manuscript and prepared the figures. All authors edited and reviewed the manuscript. Ethics approval Not applicable Consent to participate All authors approved submission Consent to publish All authors approved publication Conflicts of interest The authors declare that they have no conflict of interest Data and material 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 PXD 069032. Funding 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 of K.D. was supported by a fellowship from the French Ministry of Research (MESRI). Acknowledgements We are grateful for the Institut National de la Santé et de la Recherche Médicale INSERM), Aix-Marseille Université (AMU) and the Agence Nationale de la Recherche (ANR) for their financial support. We thank Stephanie Baulac for sharing the Lgi1 −/− mice strain [ 98 ]. Funder Information Declared Agence Nationale de la Recherche , ANR-17-CE16-0022 Institut National de la Santé et de la Recherche Médicale (INSERM) Aix-Marseille Université, https://ror.org/035xkbk20 References 1. ↵ Seagar M , Russier M , Caillard O , Maulet Y , Fronzaroli-Molinieres L , De San Feliciano M , et al. LGI1 tunes intrinsic excitability by regulating the density of axonal Kv1 channels . Proc Natl Acad Sci U S A . 2017 ; 114 : 7719 – 24 . OpenUrl Abstract / FREE Full Text 2. ↵ Extremet J , El Far O , Ankri N , Irani SR , Debanne D , Russier M . An Epitope-Specific LGI1-Autoantibody Enhances Neuronal Excitability by Modulating Kv1.1 Channel . 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Share A hyperglycosylated form of Kv 1.2 upregulated in LGI1 knockout mice Jorge Ramirez-Franco , Marion Sangiardi , Kévin Debreux , Maya Belghazi , Christian Lévêque , Michael Seagar , Oussama El Far bioRxiv 2025.10.29.685130; doi: https://doi.org/10.1101/2025.10.29.685130 Share This Article: Copy Citation Tools A hyperglycosylated form of Kv 1.2 upregulated in LGI1 knockout mice Jorge Ramirez-Franco , Marion Sangiardi , Kévin Debreux , Maya Belghazi , Christian Lévêque , Michael Seagar , Oussama El Far bioRxiv 2025.10.29.685130; doi: https://doi.org/10.1101/2025.10.29.685130 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41910) Biophysics (21436) Cancer Biology (18576) Cell Biology (25480) Clinical Trials (138) Developmental Biology (13368) Ecology (19887) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15598) Genomics (22482) Immunology (17726) Microbiology (40360) Molecular Biology (17163) Neuroscience (88534) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7637) Plant Biology (15129) Scientific Communication and Education (2045) Synthetic Biology (4290) Systems Biology (9817) Zoology (2269)