Novel post-translational modification in voltage-gated potassium channel KCNQ2

Novel post-translational modification in voltage-gated potassium channel KCNQ2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Novel post-translational modification in voltage-gated potassium channel KCNQ2 Yuichi Kimura, Hidehiko Uchiyama, Koji Masuda, Shinichi Hirose This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7965157/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract KCNQ2 is a member of the voltage-gated potassium (Kv) channel family and regulates neuronal activity through potassium ion efflux. Pathogenic variants of KCNQ2 lead to aberrant neuronal activity and cause two types of epilepsy: self-limited familial neonatal epilepsy (SLFNE) and developmental and epileptic encephalopathies (DEE). However, how these pathogenic variants influence KCNQ2 expression remains unclear. Here we show a short isoform of mouse KCNQ2 (KCNQ2 S ), whose expression levels differed significantly across variants compared with wild type, whereas those of full-length KCNQ2 (KCNQ2 F ) remained unchanged. Of particular interest, two variants at residue Y284, Y284C and Y284D, which are associated with distinct clinical phenotypes—self-limited familial neonatal epilepsy (SLFNE) and developmental and epileptic encephalopathy (DEE), respectively—exerted opposite effects on the short isoform: Y284C increased the level of KCNQ2 S , whereas Y284D decreased it compared with the wild type. As KCNQ2 S was found to be localized in the plasma membrane, it is suggested that KCNQ2 S is a post-translational product resulting from a cleavage of full-length KCNQ2. This novel post-translational cleavage generating KCNQ2 S was observed in neuronal cells and appears to be evolutionarily conserved. Although the role of this post-translational modification in epilepsy remains unknown, it may be elucidated through future studies. Health sciences/Diseases Health sciences/Neurology Biological sciences/Neuroscience Voltage-gated potassium channel KCNQ2 Kv7.2 Post-translational modification (PTM) Proteolytic cleavage Epilepsy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Voltage-gated potassium (Kv) channels are essential for maintaining the resting membrane potential and regulating neuronal excitability [1]. These ion channels consist of four voltage-sensing domains (VSDs) and two pore-forming domains and are classified into 12 subfamilies [2, 3, 4]. The human KCNQ2 ( hKCNQ2 ) gene encodes Kv7.2, a member of the Kv7 channel family. Kv7.2 forms homo- or hetero-tetramers with Kv7.3, encoded by hKCNQ3 , a known paralog of hKCNQ2 [5, 6]. Per previous studies, pathogenic variants in hKCNQ2 are associated with two distinct epilepsy syndromes—self-limited familial neonatal epilepsy (SLFNE) and developmental and epileptic encephalopathy (DEE)—due to their impact on neuronal excitability [7, 8, 9, 10]. Both conditions typically manifest within the first few days after birth; SLFNE usually resolves within weeks with a good prognosis, whereas DEE represents an intractable form of epilepsy accompanied by developmental delay [6]. Diverse pathogenic variants in hKCNQ2 , including frameshift, insertion, deletion, and single-nucleotide polymorphisms (SNPs), have been identified in affected individuals [11, 12]. Most KCNQ2 variants are associated with loss-of-function rather than gain-of-function effects [4, 13, 14, 15]. Several studies have reported that pathogenic variants of KCNQ2 can alter protein expression levels and subcellular localization in neurons [11, 16, 17]. However, the mechanisms by which these mutations affect KCNQ2 protein processing and expression remain largely unknown. To explore this issue, we analyzed the expression of mouse KCNQ2 (mKCNQ2) carrying several pathogenic variants previously identified in either self-limited familial neonatal epilepsy (SLFNE) or developmental and epileptic encephalopathy (DEE), including T274M, Y284C, Y284D, G290D, and A306T [18, 19, 20, 21]. In this study, we discovered a novel post-translational modification of KCNQ2—a proteolytic cleavage that generates a shorter isoform—and found that the proportion of this truncated form varied depending on the mutation. Results Different expression patterns of mKCNQ2 across genotypes To investigate the basic gene expression profile of mouse KCNQ2 ( mKcnq2 ), we focused on five genotypes previously reported as pathogenic mutations: Thr274Met (T274M), Tyr284Asp (Y284D) and Gly290Asp (G290D), associated with DEE; and Tyr284Cys (Y284C) and Ala306Thr (A306T), associated with SLFNE (18 – 21) . We constructed carboxyl-terminal 3xFLAG ("DYKDDDDK")-tagged mKcnq2 plasmids harboring each variant (Fig. 1 A) and examined mKCNQ2 protein expression using western blotting with an anti-FLAG antibody (Fig. 1 B). Full-length mKCNQ2 (mKCNQ2 F ) protein levels were not significantly different among the genotypes (Fig. 1 B, 1 C). However, we observed an additional low-molecular-weight band, which appeared to be missing the N-terminus, and named it short-chain mKCNQ2 (mKCNQ2 S ). Quantitative analysis showed that mKCNQ2 S expression was significantly increased in the T274M, Y284C and A306T variants and decreased in Y284D and G290D compared with that in WT (Fig. 1 B, D). Notably, Y284C and Y284D, which differ by a single amino acid substitution, exhibited opposite expression trends. Together, these results suggest that the five pathogenic SNPs affect mKCNQ2 protein expression, as compared to WT. mKCNQ2 may undergo cleavage via post-translational modification While mKCNQ2 F is known to localize to the cell membrane, the subcellular localization of mKCNQ2 S remains unclear. To address this, we separated cells into cytoplasmic and pellet fractions and performed Western blotting. Both mKCNQ2 F and mKCNQ2 S were detected in the pellet fraction (Fig. 2 A), suggesting their membrane association. The mechanism underlying mKCNQ2 S synthesis remained unclear. We therefore hypothesized that mKCNQ2 S arises either from aberrant translational initiation or from protein cleavage. To examine this hypothesis, we generated mKcnq2 plasmids bearing an amino-terminal 1xMYC tag and a carboxyl–terminal 3xFLAG tag harboring each genotype (Fig. 2 B). Western blotting with anti-Myc antibody showed two bands that yielded similar results with anti-FLAG (Fig. 2 C), we named it N-terminus short-chain mKCNQ2 (mKCNQ2 S − N ). This result supporting the hypothesis that mKCNQ2 S results from protein cleavage rather than aberrant translation initiation. Based on the possibility that mKCNQ2 undergoes proteolytic cleavage, we investigated the potential cleavage site. Western blotting using anti-Myc (N-terminal) and anti-FLAG (C-terminal) antibodies detected two distinct fragments of approximately 28 kDa and 65 kDa, respectively (Fig. 2 A, C). The 65 kDa C-terminal fragment was designated mKCNQ2 S , whereas the 28 kDa N-terminal fragment mKCNQ2 S − N corresponded to the remaining portion of the full-length protein. The combined molecular weights of these two fragments were roughly equivalent to that of mKCNQ2 F , indicating that mKCNQ2 S represents a C-terminal product generated by post-translational cleavage of mKCNQ2 F . Based on these findings, we hypothesized that the cleavage site was located within a 100–amino acid region spanning residues 151–250, which encompasses the S3 to S5 domains (Fig. 3 A). To test this, we constructed mKcnq2 plasmids carrying deletions within this region on the A306T background, which exhibits high expression of mKCNQ2 S . Among these constructs, deletion of residues 171–180 abolished mKCNQ2 S expression while preserving that of the full-length form (mKCNQ2 F ) (Fig. 3 B). This result suggested that the cleavage site is likely located around residues 171–180. Supporting this, smaller deletions of residues 171–175 or 176–180 still produced detectable mKCNQ2 S (Fig. 3 B). In silico analysis using peptide cutter software of Expasy, which is operated by the Swiss Institute of Bioinformatics ( https://web.expasy.org/peptide_cutter/ ), predicted that the 10 amino acids region (171–180) included cleavage sites for Asp-N endopeptidase and chymotrypsin. To experimentally verify this prediction, we performed alanine-scanning mutagenesis, a method in which amino acid residues within a target region are systematically replaced with alanine. Because alanine lacks bulky or charged side chains, such substitutions often disrupt protease recognition motifs and render the region resistant to enzymatic cleavage. Using this approach, we generated mKcnq2 plasmids in which the residues within the predicted cleavage region (171–180) were substituted with alanine. However, mKCNQ2 S was still detected in all mutant constructs (Fig. 3 C). Finally, protease inhibitor for serine, cysteine, aspartic proteases and aminopeptidases addition did not abolish the cleavage of mKCNQ2 (Fig. 3 D). Taken together, these results suggest that both mKCNQ2 F and mKCNQ2 S are expressed on the membrane, and that mKCNQ2 may be cleaved via a novel post-translational modification mechanism. mKCNQ3 expression pattern differs from that of mKCNQ2 hKCNQ3 forms both homo- and/or hetero-tetramers with hKCNQ2 and has been implicated in conditions including SLFNE and neurocognitive deficits [ 4 , 7 , 22 , 23 , 24 ]. However, as with hKCNQ2 , it remains unclear how specific genetic variants in hKCNQ3 gene contribute to disease pathogenesis. Given that hKCNQ3 is a paralog of hKCNQ2 , we hypothesized that mKCNQ3 may also get cleaved, similar to mKCNQ2. To verify this, we constructed carboxyl–terminally 3xFLAG-tagged mKcnq3 plasmids encoding genotypes of WT as well as the variants G311V, R331C, R331H and R331L (Fig. 4 A). Interestingly, Western blot analysis revealed that only full-length mKCNQ3 was detected (Fig. 4 B), with no evidence of a cleaved isoform. The amino acid sequence homology between the S3 domains of mKCNQ2 and mKCNQ3 was 57%, specifically 70% for the predicted cleavage site (Fig. 4 C). These results suggest that the regulatory mechanism of gene expression differs between mKCNQ2 and mKCNQ3. KCNQ2 cleavage may be regulated by post-translational modification, similar to that for mKCNQ2 While our analyses focused on mKCNQ2, it remained unclear whether human KCNQ2 is also subject to proteolytic cleavage. Few studies have focused on the molecular mechanisms regulating KCNQ2 gene expression. In order to determine whether protein cleavage of mKCNQ2 represents a mouse-specific post-translational control or an evolutionarily conserved mechanism, we constructed carboxyl-terminal 3xFLAG-tagged human KCNQ2 expression plasmids and examined KCNQ2 expression pattern (Fig. 5 A). Western blot analysis showed the presence of both full-length and cleaved KCNQ2 isoforms, and the expression patterns of individual variants mirrored those observed for mKCNQ2 (Fig. 5 B), indicating that the mechanism of KCNQ2 cleavage is conserved between human and mouse. To further investigate whether the cleavage is specific to cell type or species, we expressed hKCNQ2 in the human neuroblastoma cell line SH-SY5Y and mKCNQ2 in HEK293T cells. In both systems, we could detect cleaved KCNQ2 bands, and expression patters were similar to that presented in Fig. 1 results (Fig. 5 C, D). These findings suggest that KCNQ2 cleavage is an evolutionarily conserved post-translational mechanism that occurs not only in heterologous expression systems but also in neuronal cells. This indicates that proteolytic processing of KCNQ2 is a physiologically relevant event, rather than an artifact limited to HEK293T cells. Discussion Pathogenic variants in KCNQ2 are associated with two clinically distinct epileptic disorders—self-limited familial neonatal epilepsy (SLFNE) and developmental and epileptic encephalopathy (DEE)—yet the molecular basis underlying their phenotypic divergence remains poorly understood. While most previous studies have emphasized electrophysiological abnormalities caused by channel dysfunction, relatively little is known about how these variants influence KCNQ2 expression and post-translational regulation. In line with earlier reports [ 11 , 25 ], our study found no marked differences in total KCNQ2 mRNA or protein levels among the tested variants, indicating that endoplasmic-reticulum–associated degradation (ERAD) or calmodulin-dependent mechanisms are unlikely to explain the variant-dependent phenotypes. Instead, we identified a previously uncharacterized short isoform of KCNQ2, termed KCNQ2 S , that results from post-translational cleavage of the full-length protein (KCNQ2 F ). Although similar short bands have occasionally been observed in earlier studies [ 16 ], their significance had not been addressed. Here, we demonstrate that KCNQ2 S expression varies depending on genotype, most notably between the Y284C and Y284D variants, which are associated with SLFNE and DEE, respectively. These variants exert opposite effects on KCNQ2 S expression, suggesting a potential link between the relative abundance of KCNQ2 S and disease phenotype. Biochemical analysis revealed that KCNQ2 S corresponds to the C-terminal fragment generated by cleavage within the S3 domain of KCNQ2. The 171–180 amino acid region was found to be essential for the appearance of KCNQ2 S , implying that this segment harbors a critical cleavage site. The S3 domain plays a central role in voltage sensing through its interaction with S2 and S4 segments [ 4 , 14 , 26 , 27 ]. In the resting state, residue D172 in S3 forms electrostatic interactions with R201 or R213 in S4, contributing to channel gating. Cleavage within this region could therefore alter conformational coupling between the voltage-sensing and pore domains, potentially modulating channel function. The proteolytic mechanism responsible for generating KCNQ2 S remains to be elucidated. The fact that neither alanine-scanning mutagenesis nor protease inhibitor treatment prevented KCNQ2 S formation suggests involvement of a noncanonical cleavage process. Given that the S3 domain resides within the transmembrane region, accessibility by conventional proteases is limited. One possible explanation is the participation of intramembrane-cleaving proteases (i-CLiPs), such as γ-secretase, site-2 protease (S2P), or rhomboid proteases [ 28 , 29 , 30 , 31 ]. These evolutionarily conserved enzymes mediate regulated intramembrane proteolysis, and some act on ion channel proteins, although their specific substrate sequences remain incompletely characterized [ 32 , 33 , 34 , 35 ]. Our observations are consistent with the possibility that an i-CLiP–like mechanism contributes to KCNQ2 cleavage. The functional significance of KCNQ2 S remains speculative, but its conservation across species and occurrence in neuronal as well as non-neuronal cells suggest a physiologically relevant process. Cleavage may serve to regulate the stability, localization, or turnover of Kv7.2 channels under specific cellular conditions. The finding that the Y284C and Y284D variants—linked to opposite clinical phenotypes—induce inverse effects on KCNQ2 S expression supports the notion that dysregulation of this post-translational process could influence disease pathogenesis. Whether differences in KCNQ2 S levels directly alter channel conductance or subunit assembly remains to be clarified. In summary, our study identifies a novel post-translational cleavage of KCNQ2 within the S3 domain and demonstrates that the relative expression of the cleaved isoform KCNQ2 S varies among pathogenic variants. This cleavage process appears evolutionarily conserved and occurs in neuronal cells, implying a potential role in the physiological regulation of KCNQ2 function. Future investigations should aim to identify the responsible protease and to elucidate how altered KCNQ2 S formation contributes to epileptic phenotypes such as SLFNE and DEE. Methods Mouse Kcnq2 , Kcnq3 and human KCNQ2 gene cloning and plasmid preparation Coding sequences of mouse Kcnq2 , Kcnq3 , and human KCNQ2 were amplified by PCR using Mouse Brain QUICK-CloneTM cDNA and Human Fetal Brain QUICK-CloneTM cDNA as templates (Clontech). The amplified mKcnq2 and hKCNQ2 plasmids were constructed using the pIRES2-EGFP vector (Clontech) and the In-Fusion HD Cloning Kit (Clontech). Amino-terminal Myc and carboxyl-terminal FLAG tags were introduced using the same cloning strategy. Site-directed mutagenesis was performed using the KOD -Plus- Mutagenesis kit (TOYOBO) to generate plasmids harboring each genetic variant. Cell culture, transfection, protein extraction, and cell treatments Neuro2A and HEK293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose; Sigma) supplemented with 10% fetal bovine serum (FBS). SH-SY5Y cells were cultured in DMEM/Nutrient Mixture F-12 Ham (Sigma) supplemented with 10% FBS and MEM Non-essential Amino Acid Solution (Sigma). All cells were incubated at 37℃ in a humidified 5% CO 2 atmosphere. Transfection of cells was performed using LipofectamineTM 3000 (Thermo Fisher Scientific) with 1 µg of plasmid DNA, according to the manufacturer’s instructions. Twenty-four hours post-transfection, cells were washed with PBS. Extract and pellet fractions were separated from the cultured cells using an extraction buffer (20 mM Tris-HCl, 15 mM NaCl, and 0.1% Triton X-100). Proteins were extracted by mixing the cell lysates with SDS sample buffer and sonicating the samples. Protease inhibitors (Protease Inhibitor Cocktail; Sigma) were added to cells 30 min before transfection to prevent protein degradation. Western blot analysis, antibodies, quantification, and statistical analysis For Western blot analysis, the following antibodies were used: anti-FLAG M2 monoclonal antibody (F1804; Sigma), anti-GFP monoclonal antibody (1E4; Medical & Biological Laboratories), anti-Myc monoclonal antibody (My3; Medical & Biological Laboratories), and anti-KCNQ2 polyclonal antibody (APC-050; Alomone Labs). Phosphorylated proteins were separated using Phos-tagTM Acrylamide (Fuji Film) according to the manufacturer’s instructions. Protein detection was performed using the AI680 imaging system (Cytiva) with Immobilon Western Chemiluminescent Horseradish Peroxidase Substrate (Merck Millipore). Signal intensities were quantified using the analysis software attached to the AI680 system. Statistical analyses were performed using BellCurve for Excel (Social Survey Research Information Co., Ltd.). Declarations Acknowledgements Authors thank Akiyo Hamachi and Dr. Yoshio Misumi for technical support. Author contributions Y. K. and S. H. designed research strategies. Y. K. performed experiments, analyzed data, and wrote manuscript. Y. K., H. U., K. M. and S. H. discussed about results and solutions. All authors reviewed the manuscript. Funding This research was supported by JSPS KAKENHI (grant JP19K17314 to Y. K.) and The Epilepsy Research Foundation (grant TENKAN20003 to Y. K.). Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. 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Supplementary Files ScirepSupplementaryFigure.pdf Cite Share Download PDF Status: Published Journal Publication published 04 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 04 Dec, 2025 Reviews received at journal 03 Dec, 2025 Reviewers agreed at journal 02 Dec, 2025 Reviews received at journal 19 Nov, 2025 Reviewers agreed at journal 07 Nov, 2025 Reviewers agreed at journal 05 Nov, 2025 Reviewers agreed at journal 05 Nov, 2025 Reviewers invited by journal 05 Nov, 2025 Editor invited by journal 03 Nov, 2025 Editor assigned by journal 29 Oct, 2025 Submission checks completed at journal 29 Oct, 2025 First submitted to journal 27 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7965157","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":545086338,"identity":"b7640c95-f3da-4e7e-a938-2da22717e54c","order_by":0,"name":"Yuichi 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Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Hidehiko","middleName":"","lastName":"Uchiyama","suffix":""},{"id":545086340,"identity":"fea4e23e-81cb-4db4-a725-a56b3ac1e3fc","order_by":2,"name":"Koji Masuda","email":"","orcid":"","institution":"Tokyo University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Koji","middleName":"","lastName":"Masuda","suffix":""},{"id":545086341,"identity":"e75a9bea-e958-4e4a-b165-77b6f5ca1e93","order_by":3,"name":"Shinichi Hirose","email":"","orcid":"","institution":"Fukuoka University","correspondingAuthor":false,"prefix":"","firstName":"Shinichi","middleName":"","lastName":"Hirose","suffix":""}],"badges":[],"createdAt":"2025-10-28 12:00:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7965157/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7965157/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-42444-9","type":"published","date":"2026-03-04T15:58:39+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":96250929,"identity":"e2e0c2c7-6c7a-4626-b64e-47962a8a7cc3","added_by":"auto","created_at":"2025-11-19 07:39:08","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":609106,"visible":true,"origin":"","legend":"","description":"","filename":"ScirepManuscriptfile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7965157/v1/d28dec11e0b638c2131c6f8e.docx"},{"id":96157159,"identity":"3a745e84-447e-4ccf-9bf3-837259cce534","added_by":"auto","created_at":"2025-11-18 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08:33:53","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":85976,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7965157/v1/df43fec020b7a9ed9cdb66c3.html"},{"id":96157162,"identity":"6b1683c7-f317-47f2-98e8-c85a787c59c8","added_by":"auto","created_at":"2025-11-18 08:33:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":94695,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eShort-length mKCNQ2 expression differs significantly among genetic variants.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo analyze mKCNQ2 protein expression, we generated plasmids encoding each variant of \u003cem\u003emKcnq2\u003c/em\u003efused to a 3×FLAG- tagged carboxyl-terminal and independently expressing EGFP via an IRES sequence (A). Plasmids were transfected into Neuro 2A cells. mKCNQ2 and GFP proteins were immunoblotted with anti-FLAG and anti-GFP antibodies, respectively (B). The intensities of mKCNQ2-FLAG were measured and normalized to GFP levels, and compared with WT. Data are presented as mean ± SEM (n = 5). *p \u0026lt; 0.05, determined by Kruskal–Wallis test followed by Steel’s multiple compression test (C and D). T274M, Y284D, and G290D are pathogenic variants of the DEE, while Y284C and A306T are pathogenic variants of the SLFNE. Original blots are presented in Supplementary Figure 1.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7965157/v1/7cbdf17467d12ccbe52760dc.png"},{"id":96157158,"identity":"cfb81234-6d57-4673-a038-93244eb5b55a","added_by":"auto","created_at":"2025-11-18 08:33:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":117276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eN-terminal and C-terminal regions of mKCNQ2 express in the pellet fraction.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach \u003cem\u003emKcnq2\u003c/em\u003eplasmid was transfected into N2A cells. Whole-cell extracts were separated into soluble (extract) and pellet fractions. mKCNQ2 samples were immunoblotted with anti-FLAG and anti-GFP antibodies (A). To determine amino-terminal mKCNQ2 protein expression, we generated encoding \u003cem\u003emKcnq2\u003c/em\u003e variants fused with an N-terminal 1×Myc tag and a C-terminal 3×FLAG tag (B). Separated cell extracts were analyzed by immunoblotting with anti-Myc and anti-GFP antibodies (C). Original blots are presented in Supplementary Figure 2, 3.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7965157/v1/308cf10f25fa1f5e2794a356.png"},{"id":96250788,"identity":"d386e8af-b30d-44f2-a86f-753aa6f0e167","added_by":"auto","created_at":"2025-11-19 07:39:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":187354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of the amino acid region required for KCNQ2\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmino acid residues 151 to 250 of mKCNQ2 are shown (A). To determine which region is required for KCNQ2\u003csup\u003eS\u003c/sup\u003e expression, we generated deletion mutants lacking specific amino acids based on the A306T variant of mKCNQ2. These plasmids were transfected into Neuro2A cell and mKCNQ2 protein expression was assessed by immunoblotting with anti-FLAG antibody (B). To observe the effect of protease, any amino acid residue was replaced with alanine of mKCNQ2. The plasmids were then transfected into Neuro2A cells, and mKCNQ2 protein expression was assessed via immunoblotting with anti-FLAG antibody (C). To analyze mKCNQ2S protein expression, we added protease inhibitor 30 min before transfection in Neuro2A cells. mKCNQ2 samples were immunoblotted with anti-FLAG antibodies (D). Original blots are presented in Supplementary Figure 4, 5, 6.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7965157/v1/e7d810fe0b6a665a16333c18.png"},{"id":96157163,"identity":"f0c489ae-7db5-4364-b980-e9c799e80f89","added_by":"auto","created_at":"2025-11-18 08:33:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":87998,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eShort-length proteins are not expressed in mKCNQ3.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo analyze mKCNQ3 protein expression, we generated each plasmids encoding C-terminal 3×FLAG-tagged \u003cem\u003emKcnq3\u003c/em\u003e variants with independent EGFP expression via an IRES sequence (A). Plasmids were transfected into Neuro2A cells. mKCNQ3 and GFP proteins were immunoblotted with anti-FLAG and anti-GFP antibodies (B). Comparison of amino acid sequences of the S3 domain between mKCNQ2 and mKCNQ3. Glay highlited means predicted cleavage region (C). Original blots are presented in Supplementary Figure 7.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7965157/v1/2cfe34ef2d5023c33b25f23c.png"},{"id":96251993,"identity":"04bd9a90-085a-4b64-81b7-a0f7988a7775","added_by":"auto","created_at":"2025-11-19 07:40:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":152933,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eShort-length KCNQ2 isoforms are evolutionarily conserved.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo analyze hKCNQ2 protein expression, we generated plasmids encoding each variant of \u003cem\u003ehKcnq2\u003c/em\u003efused to a 3×FLAG-tagged carboxyl-terminal and independently expressing EGFP via an IRES sequence (A). Plasmids were transfected into Neuro2A and SH-SY5Y cells. hKCNQ2 and GFP proteins were immunoblotted with anti-FLAG and anti-GFP antibodies (B and C). \u003cem\u003emKcnq2\u003c/em\u003e plasmids were also transfected in HEK293T cells, and mKCNQ2 and GFP proteins were detected using immunoblotting with anti-FLAG and anti-GFP antibodies (D). Original blots are presented in Supplementary Figure 8, 9, 10.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7965157/v1/0d757a97b39ef3d8b50b1c99.png"},{"id":104250813,"identity":"54c1d112-33e5-48ce-aa1a-c80106608310","added_by":"auto","created_at":"2026-03-09 16:09:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1455123,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7965157/v1/fcf4d2cd-6467-420e-8796-8978e92648aa.pdf"},{"id":96157160,"identity":"baf5864f-631e-4532-8882-7ba07e8caeac","added_by":"auto","created_at":"2025-11-18 08:33:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":585629,"visible":true,"origin":"","legend":"","description":"","filename":"ScirepSupplementaryFigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7965157/v1/8802501be3a823e47ca10e01.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Novel post-translational modification in voltage-gated potassium channel KCNQ2","fulltext":[{"header":"Introduction","content":"\u003cp\u003eVoltage-gated potassium (Kv) channels are essential for maintaining the resting membrane potential and regulating neuronal excitability [1]. These ion channels consist of four voltage-sensing domains (VSDs) and two pore-forming domains and are classified into 12 subfamilies [2, 3, 4]. The human \u003cem\u003eKCNQ2\u003c/em\u003e (\u003cem\u003ehKCNQ2\u003c/em\u003e) gene encodes Kv7.2, a member of the Kv7 channel family. Kv7.2 forms homo- or hetero-tetramers with Kv7.3, encoded by \u003cem\u003ehKCNQ3\u003c/em\u003e, a known paralog of \u003cem\u003ehKCNQ2\u003c/em\u003e [5, 6].\u003c/p\u003e\n\u003cp\u003ePer previous studies, pathogenic variants in \u003cem\u003ehKCNQ2\u003c/em\u003e are associated with two distinct epilepsy syndromes—self-limited familial neonatal epilepsy (SLFNE) and developmental and epileptic encephalopathy (DEE)—due to their impact on neuronal excitability [7, 8, 9, 10]. Both conditions typically manifest within the first few days after birth; SLFNE usually resolves within weeks with a good prognosis, whereas DEE represents an intractable form of epilepsy accompanied by developmental delay [6]. Diverse pathogenic variants in \u003cem\u003ehKCNQ2\u003c/em\u003e, including frameshift, insertion, deletion, and single-nucleotide polymorphisms (SNPs), have been identified in affected individuals [11, 12]. Most \u003cem\u003eKCNQ2\u003c/em\u003e variants are associated with loss-of-function rather than gain-of-function effects [4, 13, 14, 15].\u003c/p\u003e\n\u003cp\u003eSeveral studies have reported that pathogenic variants of \u003cem\u003eKCNQ2\u003c/em\u003e can alter protein expression levels and subcellular localization in neurons [11, 16, 17]. However, the mechanisms by which these mutations affect KCNQ2 protein processing and expression remain largely unknown. To explore this issue, we analyzed the expression of mouse KCNQ2 (mKCNQ2) carrying several pathogenic variants previously identified in either self-limited familial neonatal epilepsy (SLFNE) or developmental and epileptic encephalopathy (DEE), including T274M, Y284C, Y284D, G290D, and A306T [18, 19, 20, 21].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we discovered a novel post-translational modification of KCNQ2—a proteolytic cleavage that generates a shorter isoform—and found that the proportion of this truncated form varied depending on the mutation.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\u003ch2\u003eDifferent expression patterns of mKCNQ2 across genotypes\u003c/h2\u003e\u003cp\u003eTo investigate the basic gene expression profile of mouse \u003cem\u003eKCNQ2\u003c/em\u003e (\u003cem\u003emKcnq2\u003c/em\u003e), we focused on five genotypes previously reported as pathogenic mutations: Thr274Met (T274M), Tyr284Asp (Y284D) and Gly290Asp (G290D), associated with DEE; and Tyr284Cys (Y284C) and Ala306Thr (A306T), associated with SLFNE \u003csup\u003e(18 \u0026ndash; 21)\u003c/sup\u003e. We constructed carboxyl-terminal 3xFLAG (\"DYKDDDDK\")-tagged \u003cem\u003emKcnq2\u003c/em\u003e plasmids harboring each variant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and examined mKCNQ2 protein expression using western blotting with an anti-FLAG antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Full-length mKCNQ2 (mKCNQ2\u003csup\u003eF\u003c/sup\u003e) protein levels were not significantly different among the genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). However, we observed an additional low-molecular-weight band, which appeared to be missing the N-terminus, and named it short-chain mKCNQ2 (mKCNQ2\u003csup\u003eS\u003c/sup\u003e). Quantitative analysis showed that mKCNQ2\u003csup\u003eS\u003c/sup\u003e expression was significantly increased in the T274M, Y284C and A306T variants and decreased in Y284D and G290D compared with that in WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, D). Notably, Y284C and Y284D, which differ by a single amino acid substitution, exhibited opposite expression trends. Together, these results suggest that the five pathogenic SNPs affect mKCNQ2 protein expression, as compared to WT.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003emKCNQ2 may undergo cleavage via post-translational modification\u003c/h2\u003e\u003cp\u003eWhile mKCNQ2\u003csup\u003eF\u003c/sup\u003e is known to localize to the cell membrane, the subcellular localization of mKCNQ2\u003csup\u003eS\u003c/sup\u003e remains unclear. To address this, we separated cells into cytoplasmic and pellet fractions and performed Western blotting. Both mKCNQ2\u003csup\u003eF\u003c/sup\u003e and mKCNQ2\u003csup\u003eS\u003c/sup\u003e were detected in the pellet fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), suggesting their membrane association. The mechanism underlying mKCNQ2\u003csup\u003eS\u003c/sup\u003e synthesis remained unclear. We therefore hypothesized that mKCNQ2\u003csup\u003eS\u003c/sup\u003e arises either from aberrant translational initiation or from protein cleavage. To examine this hypothesis, we generated \u003cem\u003emKcnq2\u003c/em\u003e plasmids bearing an amino-terminal 1xMYC tag and a carboxyl\u0026ndash;terminal 3xFLAG tag harboring each genotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Western blotting with anti-Myc antibody showed two bands that yielded similar results with anti-FLAG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), we named it N-terminus short-chain mKCNQ2 (mKCNQ2\u003csup\u003eS\u0026thinsp;\u0026minus;\u0026thinsp;N\u003c/sup\u003e). This result supporting the hypothesis that mKCNQ2\u003csup\u003eS\u003c/sup\u003e results from protein cleavage rather than aberrant translation initiation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBased on the possibility that mKCNQ2 undergoes proteolytic cleavage, we investigated the potential cleavage site. Western blotting using anti-Myc (N-terminal) and anti-FLAG (C-terminal) antibodies detected two distinct fragments of approximately 28 kDa and 65 kDa, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, C). The 65 kDa C-terminal fragment was designated mKCNQ2\u003csup\u003eS\u003c/sup\u003e, whereas the 28 kDa N-terminal fragment mKCNQ2\u003csup\u003eS\u0026thinsp;\u0026minus;\u0026thinsp;N\u003c/sup\u003e corresponded to the remaining portion of the full-length protein. The combined molecular weights of these two fragments were roughly equivalent to that of mKCNQ2\u003csup\u003eF\u003c/sup\u003e, indicating that mKCNQ2\u003csup\u003eS\u003c/sup\u003e represents a C-terminal product generated by post-translational cleavage of mKCNQ2\u003csup\u003eF\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBased on these findings, we hypothesized that the cleavage site was located within a 100\u0026ndash;amino acid region spanning residues 151\u0026ndash;250, which encompasses the S3 to S5 domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To test this, we constructed \u003cem\u003emKcnq2\u003c/em\u003e plasmids carrying deletions within this region on the A306T background, which exhibits high expression of mKCNQ2\u003csup\u003eS\u003c/sup\u003e. Among these constructs, deletion of residues 171\u0026ndash;180 abolished mKCNQ2\u003csup\u003eS\u003c/sup\u003e expression while preserving that of the full-length form (mKCNQ2\u003csup\u003eF\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). This result suggested that the cleavage site is likely located around residues 171\u0026ndash;180. Supporting this, smaller deletions of residues 171\u0026ndash;175 or 176\u0026ndash;180 still produced detectable mKCNQ2\u003csup\u003eS\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn silico analysis using peptide cutter software of Expasy, which is operated by the Swiss Institute of Bioinformatics (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/peptide_cutter/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/peptide_cutter/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), predicted that the 10 amino acids region (171\u0026ndash;180) included cleavage sites for Asp-N endopeptidase and chymotrypsin. To experimentally verify this prediction, we performed alanine-scanning mutagenesis, a method in which amino acid residues within a target region are systematically replaced with alanine. Because alanine lacks bulky or charged side chains, such substitutions often disrupt protease recognition motifs and render the region resistant to enzymatic cleavage. Using this approach, we generated \u003cem\u003emKcnq2\u003c/em\u003e plasmids in which the residues within the predicted cleavage region (171\u0026ndash;180) were substituted with alanine. However, mKCNQ2\u003csup\u003eS\u003c/sup\u003e was still detected in all mutant constructs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Finally, protease inhibitor for serine, cysteine, aspartic proteases and aminopeptidases addition did not abolish the cleavage of mKCNQ2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eTaken together, these results suggest that both mKCNQ2\u003csup\u003eF\u003c/sup\u003e and mKCNQ2\u003csup\u003eS\u003c/sup\u003e are expressed on the membrane, and that mKCNQ2 may be cleaved via a novel post-translational modification mechanism.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003emKCNQ3 expression pattern differs from that of mKCNQ2\u003c/h3\u003e\n\u003cp\u003ehKCNQ3 forms both homo- and/or hetero-tetramers with hKCNQ2 and has been implicated in conditions including SLFNE and neurocognitive deficits [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, as with \u003cem\u003ehKCNQ2\u003c/em\u003e, it remains unclear how specific genetic variants in \u003cem\u003ehKCNQ3\u003c/em\u003e gene contribute to disease pathogenesis. Given that \u003cem\u003ehKCNQ3\u003c/em\u003e is a paralog of \u003cem\u003ehKCNQ2\u003c/em\u003e, we hypothesized that mKCNQ3 may also get cleaved, similar to mKCNQ2. To verify this, we constructed carboxyl\u0026ndash;terminally 3xFLAG-tagged \u003cem\u003emKcnq3\u003c/em\u003e plasmids encoding genotypes of WT as well as the variants G311V, R331C, R331H and R331L (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Interestingly, Western blot analysis revealed that only full-length mKCNQ3 was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), with no evidence of a cleaved isoform. The amino acid sequence homology between the S3 domains of mKCNQ2 and mKCNQ3 was 57%, specifically 70% for the predicted cleavage site (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These results suggest that the regulatory mechanism of gene expression differs between mKCNQ2 and mKCNQ3.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eKCNQ2 cleavage may be regulated by post-translational modification, similar to that for mKCNQ2\u003c/h3\u003e\n\u003cp\u003eWhile our analyses focused on mKCNQ2, it remained unclear whether human KCNQ2 is also subject to proteolytic cleavage. Few studies have focused on the molecular mechanisms regulating KCNQ2 gene expression. In order to determine whether protein cleavage of mKCNQ2 represents a mouse-specific post-translational control or an evolutionarily conserved mechanism, we constructed carboxyl-terminal 3xFLAG-tagged human KCNQ2 expression plasmids and examined KCNQ2 expression pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Western blot analysis showed the presence of both full-length and cleaved KCNQ2 isoforms, and the expression patterns of individual variants mirrored those observed for mKCNQ2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating that the mechanism of KCNQ2 cleavage is conserved between human and mouse. To further investigate whether the cleavage is specific to cell type or species, we expressed hKCNQ2 in the human neuroblastoma cell line SH-SY5Y and mKCNQ2 in HEK293T cells. In both systems, we could detect cleaved KCNQ2 bands, and expression patters were similar to that presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese findings suggest that KCNQ2 cleavage is an evolutionarily conserved post-translational mechanism that occurs not only in heterologous expression systems but also in neuronal cells. This indicates that proteolytic processing of KCNQ2 is a physiologically relevant event, rather than an artifact limited to HEK293T cells.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePathogenic variants in KCNQ2 are associated with two clinically distinct epileptic disorders\u0026mdash;self-limited familial neonatal epilepsy (SLFNE) and developmental and epileptic encephalopathy (DEE)\u0026mdash;yet the molecular basis underlying their phenotypic divergence remains poorly understood. While most previous studies have emphasized electrophysiological abnormalities caused by channel dysfunction, relatively little is known about how these variants influence KCNQ2 expression and post-translational regulation.\u003c/p\u003e\u003cp\u003eIn line with earlier reports [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], our study found no marked differences in total KCNQ2 mRNA or protein levels among the tested variants, indicating that endoplasmic-reticulum\u0026ndash;associated degradation (ERAD) or calmodulin-dependent mechanisms are unlikely to explain the variant-dependent phenotypes. Instead, we identified a previously uncharacterized short isoform of KCNQ2, termed KCNQ2\u003csup\u003eS\u003c/sup\u003e, that results from post-translational cleavage of the full-length protein (KCNQ2\u003csup\u003eF\u003c/sup\u003e). Although similar short bands have occasionally been observed in earlier studies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], their significance had not been addressed. Here, we demonstrate that KCNQ2\u003csup\u003eS\u003c/sup\u003e expression varies depending on genotype, most notably between the Y284C and Y284D variants, which are associated with SLFNE and DEE, respectively. These variants exert opposite effects on KCNQ2\u003csup\u003eS\u003c/sup\u003e expression, suggesting a potential link between the relative abundance of KCNQ2\u003csup\u003eS\u003c/sup\u003e and disease phenotype.\u003c/p\u003e\u003cp\u003eBiochemical analysis revealed that KCNQ2\u003csup\u003eS\u003c/sup\u003e corresponds to the C-terminal fragment generated by cleavage within the S3 domain of KCNQ2. The 171\u0026ndash;180 amino acid region was found to be essential for the appearance of KCNQ2\u003csup\u003eS\u003c/sup\u003e, implying that this segment harbors a critical cleavage site. The S3 domain plays a central role in voltage sensing through its interaction with S2 and S4 segments [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In the resting state, residue D172 in S3 forms electrostatic interactions with R201 or R213 in S4, contributing to channel gating. Cleavage within this region could therefore alter conformational coupling between the voltage-sensing and pore domains, potentially modulating channel function.\u003c/p\u003e\u003cp\u003eThe proteolytic mechanism responsible for generating KCNQ2\u003csup\u003eS\u003c/sup\u003e remains to be elucidated. The fact that neither alanine-scanning mutagenesis nor protease inhibitor treatment prevented KCNQ2\u003csup\u003eS\u003c/sup\u003e formation suggests involvement of a noncanonical cleavage process. Given that the S3 domain resides within the transmembrane region, accessibility by conventional proteases is limited. One possible explanation is the participation of intramembrane-cleaving proteases (i-CLiPs), such as γ-secretase, site-2 protease (S2P), or rhomboid proteases [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These evolutionarily conserved enzymes mediate regulated intramembrane proteolysis, and some act on ion channel proteins, although their specific substrate sequences remain incompletely characterized [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Our observations are consistent with the possibility that an i-CLiP\u0026ndash;like mechanism contributes to KCNQ2 cleavage.\u003c/p\u003e\u003cp\u003eThe functional significance of KCNQ2\u003csup\u003eS\u003c/sup\u003e remains speculative, but its conservation across species and occurrence in neuronal as well as non-neuronal cells suggest a physiologically relevant process. Cleavage may serve to regulate the stability, localization, or turnover of Kv7.2 channels under specific cellular conditions. The finding that the Y284C and Y284D variants\u0026mdash;linked to opposite clinical phenotypes\u0026mdash;induce inverse effects on KCNQ2\u003csup\u003eS\u003c/sup\u003e expression supports the notion that dysregulation of this post-translational process could influence disease pathogenesis. Whether differences in KCNQ2\u003csup\u003eS\u003c/sup\u003e levels directly alter channel conductance or subunit assembly remains to be clarified.\u003c/p\u003e\u003cp\u003eIn summary, our study identifies a novel post-translational cleavage of KCNQ2 within the S3 domain and demonstrates that the relative expression of the cleaved isoform KCNQ2\u003csup\u003eS\u003c/sup\u003e varies among pathogenic variants. This cleavage process appears evolutionarily conserved and occurs in neuronal cells, implying a potential role in the physiological regulation of KCNQ2 function. Future investigations should aim to identify the responsible protease and to elucidate how altered KCNQ2\u003csup\u003eS\u003c/sup\u003e formation contributes to epileptic phenotypes such as SLFNE and DEE.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eMouse\u003c/b\u003e \u003cb\u003eKcnq2\u003c/b\u003e, \u003cb\u003eKcnq3\u003c/b\u003e \u003cb\u003eand human\u003c/b\u003e \u003cb\u003eKCNQ2\u003c/b\u003e \u003cb\u003egene cloning and plasmid preparation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCoding sequences of mouse \u003cem\u003eKcnq2\u003c/em\u003e, \u003cem\u003eKcnq3\u003c/em\u003e, and human \u003cem\u003eKCNQ2\u003c/em\u003e were amplified by PCR using Mouse Brain QUICK-CloneTM cDNA and Human Fetal Brain QUICK-CloneTM cDNA as templates (Clontech). The amplified \u003cem\u003emKcnq2\u003c/em\u003e and \u003cem\u003ehKCNQ2\u003c/em\u003e plasmids were constructed using the pIRES2-EGFP vector (Clontech) and the In-Fusion HD Cloning Kit (Clontech). Amino-terminal Myc and carboxyl-terminal FLAG tags were introduced using the same cloning strategy. Site-directed mutagenesis was performed using the KOD -Plus- Mutagenesis kit (TOYOBO) to generate plasmids harboring each genetic variant.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCell culture, transfection, protein extraction, and cell treatments\u003c/h2\u003e\u003cp\u003eNeuro2A and HEK293T cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM, high glucose; Sigma) supplemented with 10% fetal bovine serum (FBS). SH-SY5Y cells were cultured in DMEM/Nutrient Mixture F-12 Ham (Sigma) supplemented with 10% FBS and MEM Non-essential Amino Acid Solution (Sigma). All cells were incubated at 37℃ in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Transfection of cells was performed using LipofectamineTM 3000 (Thermo Fisher Scientific) with 1 \u0026micro;g of plasmid DNA, according to the manufacturer\u0026rsquo;s instructions. Twenty-four hours post-transfection, cells were washed with PBS. Extract and pellet fractions were separated from the cultured cells using an extraction buffer (20 mM Tris-HCl, 15 mM NaCl, and 0.1% Triton X-100). Proteins were extracted by mixing the cell lysates with SDS sample buffer and sonicating the samples. Protease inhibitors (Protease Inhibitor Cocktail; Sigma) were added to cells 30 min before transfection to prevent protein degradation.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eWestern blot analysis, antibodies, quantification, and statistical analysis\u003c/h3\u003e\n\u003cp\u003eFor Western blot analysis, the following antibodies were used: anti-FLAG M2 monoclonal antibody (F1804; Sigma), anti-GFP monoclonal antibody (1E4; Medical \u0026amp; Biological Laboratories), anti-Myc monoclonal antibody (My3; Medical \u0026amp; Biological Laboratories), and anti-KCNQ2 polyclonal antibody (APC-050; Alomone Labs). Phosphorylated proteins were separated using Phos-tagTM Acrylamide (Fuji Film) according to the manufacturer\u0026rsquo;s instructions. Protein detection was performed using the AI680 imaging system (Cytiva) with Immobilon Western Chemiluminescent Horseradish Peroxidase Substrate (Merck Millipore). Signal intensities were quantified using the analysis software attached to the AI680 system. Statistical analyses were performed using BellCurve for Excel (Social Survey Research Information Co., Ltd.).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Authors thank Akiyo Hamachi and Dr. Yoshio Misumi for technical support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Y. K. and S. H. designed research strategies. Y. K. performed experiments, analyzed data, and wrote manuscript. Y. K., H. U., K. M. and S. H. discussed about results and solutions. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by JSPS KAKENHI (grant JP19K17314 to Y. K.) and The Epilepsy Research Foundation (grant TENKAN20003 to Y. K.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interest regarding the contents of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWang, H. 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(2016) Why cells need intramembrane proteases\u0026ndash;a mechanistic perspective. \u003cem\u003eFEBS J.\u003c/em\u003e\u003cstrong\u003e283\u003c/strong\u003e, 1837-1845.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Voltage-gated potassium channel, KCNQ2, Kv7.2, Post-translational modification (PTM), Proteolytic cleavage, Epilepsy","lastPublishedDoi":"10.21203/rs.3.rs-7965157/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7965157/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eKCNQ2 is a member of the voltage-gated potassium (Kv) channel family and regulates neuronal activity through potassium ion efflux. Pathogenic variants of KCNQ2 lead to aberrant neuronal activity and cause two types of epilepsy: self-limited familial neonatal epilepsy (SLFNE) and developmental and epileptic encephalopathies (DEE). However, how these pathogenic variants influence KCNQ2 expression remains unclear. Here we show a short isoform of mouse KCNQ2 (KCNQ2\u003csup\u003eS\u003c/sup\u003e), whose expression levels differed significantly across variants compared with wild type, whereas those of full-length KCNQ2 (KCNQ2\u003csup\u003eF\u003c/sup\u003e) remained unchanged. Of particular interest, two variants at residue Y284, Y284C and Y284D, which are associated with distinct clinical phenotypes—self-limited familial neonatal epilepsy (SLFNE) and developmental and epileptic encephalopathy (DEE), respectively—exerted opposite effects on the short isoform: Y284C increased the level of KCNQ2\u003csup\u003eS\u003c/sup\u003e, whereas Y284D decreased it compared with the wild type. As KCNQ2\u003csup\u003eS\u003c/sup\u003e was found to be localized in the plasma membrane, it is suggested that KCNQ2\u003csup\u003eS\u003c/sup\u003e is a post-translational product resulting from a cleavage of full-length KCNQ2. This novel post-translational cleavage generating KCNQ2\u003csup\u003eS\u003c/sup\u003e was observed in neuronal cells and appears to be evolutionarily conserved. Although the role of this post-translational modification in epilepsy remains unknown, it may be elucidated through future studies. \u003c/p\u003e","manuscriptTitle":"Novel post-translational modification in voltage-gated potassium channel KCNQ2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-18 08:33:48","doi":"10.21203/rs.3.rs-7965157/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-04T10:56:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-03T19:14:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7103494513882209716066795387933074937","date":"2025-12-02T08:07:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-19T18:17:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"314918425096068228437688609949016670906","date":"2025-11-07T19:59:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"245617618746463353068118561893613706097","date":"2025-11-06T01:56:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"276335173001444857008306407664384763282","date":"2025-11-05T19:35:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-05T18:43:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-03T10:25:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-30T02:03:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-30T02:03:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-28T03:26:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"336605a0-3473-441f-8648-0408979fc7f7","owner":[],"postedDate":"November 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":57995888,"name":"Health sciences/Diseases"},{"id":57995889,"name":"Health sciences/Neurology"},{"id":57995890,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-03-09T16:04:58+00:00","versionOfRecord":{"articleIdentity":"rs-7965157","link":"https://doi.org/10.1038/s41598-026-42444-9","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-03-04 15:58:39","publishedOnDateReadable":"March 4th, 2026"},"versionCreatedAt":"2025-11-18 08:33:48","video":"","vorDoi":"10.1038/s41598-026-42444-9","vorDoiUrl":"https://doi.org/10.1038/s41598-026-42444-9","workflowStages":[]},"version":"v1","identity":"rs-7965157","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7965157","identity":"rs-7965157","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}