KCNMA1 genotype-phenotype correlation and functional validation of novel sites

KCNMA1 genotype-phenotype correlation and functional validation of novel sites | 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 Research Article KCNMA1 genotype-phenotype correlation and functional validation of novel sites Shimin Zhang, Xiao Xiao, Yanjun Yi, Hao Zheng, Li Cheng, Yue Hu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9206140/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Objective: To analyze genotype-phenotype correlations in patients with KCNMA1 variants and functionally characterize two unreported missense variants: c.853A>T (p.I285F) and c.3196G>A (p.G1066R). Methods: We retrospectively reviewed clinical data from six pediatric patients with KCNMA1 variants. Protein structural modeling and cross-species conservation analysis were performed. HEK293T cells were transfected with wild-type or mutant BK channel constructs. Channel function was assessed using whole-cell patch-clamp electrophysiology to measure current amplitude, current density, and voltage/Ca²⁺sensitivity. Protein expression was evaluated by Western blot analysis of total and membrane fractions. Results: (1) Five patients presented with epileptic seizures, including myoclonic, focal motor/non-motor, generalized tonic-clonic, and atonic seizures; two exhibited drug resistance. Paroxysmal nonkinesigenic dyskinesia (PNKD) was observed in two patients. All five patients with epilepsy demonstrated language impairment or global developmental delay. (2) The residues affected by the mutations, I285 and G1066, are highly conserved across species. Protein three-dimensional structure predictions revealed alterations in the number and distance of hydrogen bonds for both mutations. (3) Under low Ca²⁺ (1 μM), the p.I285F variant significantly reduced BK current amplitude (p < 0.05) and current density across the 50–150 mV range. At high Ca²⁺ (10 μM), current density was increased in the hyperpolarized range (-50 to 0 mV) compared to wild-type. The half-activation voltage (V₁/₂ ) was significantly left-shifted at 1 μM Ca²⁺ (p < 0.05), while the slope factor (k) remained unchanged. (4) At both 1 μM and 10 μM Ca²⁺, p.G1066R did not alter BK current amplitude. However, at 10 μM Ca²⁺, current density at 0 mV was significantly lower than wild-type (p < 0.05). The I-V curve was right-shifted, while V₁/₂ and k values remained unchanged. (5) Total protein levels of both p.I285F and p.G1066R were significantly lower than wild-type (p 0.05). Conclusion: KCNMA1 -related disorders commonly present with epilepsy, developmental delay, and PNKD3. p.I285F and p.G1066R are loss-of-function variants, impairing channel activity through reduced current amplitude, altered voltage sensitivity, and lower total protein expression, supporting their pathogenicity. KCNMA1 BK channel pathogenic variants genotype-phenotype correlation Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 12 Apr, 2026 Reviewers invited by journal 29 Mar, 2026 Editor assigned by journal 24 Mar, 2026 Submission checks completed at journal 24 Mar, 2026 First submitted to journal 23 Mar, 2026 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-9206140","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":614835762,"identity":"816d5e85-be43-48a4-89e6-f37930c44597","order_by":0,"name":"Shimin Zhang","email":"","orcid":"","institution":"Children’s Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shimin","middleName":"","lastName":"Zhang","suffix":""},{"id":614835763,"identity":"50196fef-1d00-419a-9828-75cdfedc2340","order_by":1,"name":"Xiao Xiao","email":"","orcid":"","institution":"Children’s Hospital of Chongqing Medical 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03:24:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9206140/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9206140/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106093830,"identity":"69320f69-1d77-4e57-8b85-75c404e2c01e","added_by":"auto","created_at":"2026-04-03 11:39:25","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":705661,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9206140/v1_covered_79897006-e0c2-489c-a392-e5ec4e180826.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"KCNMA1 genotype-phenotype correlation and functional validation of novel sites","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"KCNMA1, BK channel, pathogenic variants, genotype-phenotype correlation","lastPublishedDoi":"10.21203/rs.3.rs-9206140/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9206140/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003eTo analyze genotype-phenotype correlations in patients with \u003cem\u003eKCNMA1\u003c/em\u003evariants and functionally characterize two unreported missense variants: c.853A\u0026gt;T (p.I285F) and c.3196G\u0026gt;A (p.G1066R).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e We retrospectively reviewed clinical data from six pediatric patients with \u003cem\u003eKCNMA1\u003c/em\u003e variants. Protein structural modeling and cross-species conservation analysis were performed. HEK293T cells were transfected with wild-type or mutant BK channel constructs. Channel function was assessed using whole-cell patch-clamp electrophysiology to measure current amplitude, current density, and voltage/Ca²⁺sensitivity. Protein expression was evaluated by Western blot analysis of total and membrane fractions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(1) Five patients presented with epileptic seizures, including myoclonic, focal motor/non-motor, generalized tonic-clonic, and atonic seizures; two exhibited drug resistance. Paroxysmal nonkinesigenic dyskinesia (PNKD) was observed in two patients. All five patients with epilepsy demonstrated language impairment or global developmental delay.\u003c/p\u003e\n\u003cp\u003e(2) The residues affected by the mutations, I285 and G1066, are highly conserved across species. Protein three-dimensional structure predictions revealed alterations in the number and distance of hydrogen bonds for both mutations.\u003c/p\u003e\n\u003cp\u003e(3) Under low Ca²⁺ (1 μM), the p.I285F variant significantly reduced BK current amplitude (p \u0026lt; 0.05) and current density across the 50–150 mV range. At high Ca²⁺ (10 μM), current density was increased in the hyperpolarized range (-50 to 0 mV) compared to wild-type. The half-activation voltage (V₁/₂ ) was significantly left-shifted at 1 μM Ca²⁺ (p \u0026lt; 0.05), while the slope factor (k) remained unchanged.\u003c/p\u003e\n\u003cp\u003e(4) At both 1 μM and 10 μM Ca²⁺, p.G1066R did not alter BK current amplitude. However, at 10 μM Ca²⁺, current density at 0 mV was significantly lower than wild-type (p \u0026lt; 0.05). The I-V curve was right-shifted, while V₁/₂ and k values remained unchanged.\u003c/p\u003e\n\u003cp\u003e(5) Total protein levels of both p.I285F and p.G1066R were significantly lower than wild-type (p \u0026lt; 0.05), whereas membrane protein expression did not differ significantly (p \u0026gt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e \u003cem\u003eKCNMA1\u003c/em\u003e-related disorders commonly present with epilepsy, developmental delay, and PNKD3. p.I285F and p.G1066R are loss-of-function variants, impairing channel activity through reduced current amplitude, altered voltage sensitivity, and lower total protein expression, supporting their pathogenicity.\u003c/p\u003e","manuscriptTitle":"KCNMA1 genotype-phenotype correlation and functional validation of novel sites","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-02 13:36:37","doi":"10.21203/rs.3.rs-9206140/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"167202375247252809199740673931619856690","date":"2026-04-13T00:55:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-29T19:52:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-24T16:21:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-24T14:19:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Neurochemical Research","date":"2026-03-24T03:11:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f739475d-6eb9-4c0b-8228-0a89df169bbd","owner":[],"postedDate":"April 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-02T13:36:37+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-02 13:36:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9206140","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9206140","identity":"rs-9206140","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}