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Changes in the Kv1.3 expression are associated with numerous autoimmune and neurological disorders. In this study, Vm24, a potent and selective Kv1.3 inhibitor peptide isolated from scorpion venom, was genetically linked to mCherry, a red fluorescent protein characterized by high photostability, maturation, and tagging tolerance. The recombinant fusion protein (mCherry-Vm24) was expressed utilizing the Pichia pastoris expression system. Following protein purification using affinity chromatography and size-exclusion HPLC, the production of the correct fusion-peptide was confirmed using native PAGE, mass spectrometry, spectrophotometry, and electrophysiology. mCherry-Vm24 showed \u223c90-fold lower, albeit still picomolar (Kd = 280 pM), affinity for Kv1.3 as compared the native Vm24, but retained its high Kv1.3 selectivity and practically irreversible binding to Kv1.3. Confocal microscopy and flow cytometry reported that CHO cells expressing hKv1.3 channels were specifically labeled with mCherry-Vm24. Biodistribution of the fusion protein in mice showed the highest fluorescence signal in the kidneys and a specific increase of the mCherry-Vm24 signal in the spleen over nonconjugated mCherry. These unique properties make the mCherry-Vm24 construct suitable for visualization of Kv1.3 both in vitro and in vivo.<\/jats:p>","DOI":"10.1021\/acsomega.5c10479","type":"journal-article","created":{"date-parts":[[2026,2,27]],"date-time":"2026-02-27T00:43:18Z","timestamp":1772152998000},"page":"14681-14694","source":"Crossref","is-referenced-by-count":0,"title":["A Fluorescent Protein-Tagged\nScorpion Toxin with Picomolar\nAffinity for the Kv1.3 K\n                    <b>+<\/b>\n                    Channel"],"prefix":"10.1021","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2783-8044","authenticated-orcid":true,"given":"Jesu\u0301s","family":"Borrego","sequence":"first","affiliation":[{"name":"University of Debrecen , , 1. Egyetem Ter , ,","place":["Debrecen, Hungary, H-4032"]},{"name":"HUN-REN-UD Cell Biology and Signaling Research Group , 1. Egyetem Ter , ,","place":["Debrecen, Hungary, H-4032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3849-6219","authenticated-orcid":true,"given":"Muhammad Umair","family":"Naseem","sequence":"additional","affiliation":[{"name":"University of Debrecen , , 1. Egyetem Ter , ,","place":["Debrecen, Hungary, H-4032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-5946-8934","authenticated-orcid":true,"given":"Amna","family":"Al Olaimi","sequence":"additional","affiliation":[{"name":"University of Debrecen , , 1. Egyetem Ter , ,","place":["Debrecen, Hungary, H-4032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0438-4211","authenticated-orcid":true,"given":"Eva","family":"Korpos","sequence":"additional","affiliation":[{"name":"University of Debrecen , , 1. Egyetem Ter , ,","place":["Debrecen, Hungary, H-4032"]},{"name":"HUN-REN-UD Cell Biology and Signaling Research Group , 1. Egyetem Ter , ,","place":["Debrecen, Hungary, H-4032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3727-6129","authenticated-orcid":true,"given":"Arpad","family":"Szoor","sequence":"additional","affiliation":[{"name":"University of Debrecen , , 1. Egyetem Ter , ,","place":["Debrecen, Hungary, H-4032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8735-6920","authenticated-orcid":true,"given":"Gyula","family":"Batta","sequence":"additional","affiliation":[{"name":"University of Debrecen , , 1. Egyetem Ter , ,","place":["Debrecen, Hungary, H-4032"]},{"name":"University of Debrecen , , 1. Egyetem Ter , ,","place":["Debrecen, Hungary, H-4032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6227-3301","authenticated-orcid":true,"given":"Gyorgy","family":"Panyi","sequence":"additional","affiliation":[{"name":"University of Debrecen , , 1. Egyetem Ter , ,","place":["Debrecen, Hungary, H-4032"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,2,26]]},"reference":[{"issue":"1","key":"2026081508081860600_cit1","doi-asserted-by":"publisher","first-page":"C27","DOI":"10.1152\/ajpcell.00136.2017","article-title":"The secret life of ion channels:\nKv1.3 potassium channels and proliferation","volume":"314","author":"Perez-Garcia","year":"2018","journal-title":"Am. J. Physiol. Cell Physiol."},{"issue":"1","key":"2026081508081860600_cit2","doi-asserted-by":"publisher","first-page":"45","DOI":"10.1186\/s12964-018-0257-7","article-title":"Kv1.3 channel\nblockade with the Vm24 scorpion toxin attenuates the CD4+ effector\nmemory T cell response to TCR stimulation","volume":"16","author":"Veytia-Bucheli","year":"2018","journal-title":"Cell\nCommun. Signal."},{"key":"2026081508081860600_cit3","doi-asserted-by":"publisher","first-page":"1393","DOI":"10.3389\/fnins.2019.01393","article-title":"Kv1.3 Channel\nas a Key Therapeutic Target for Neuroinflammatory Diseases: State\nof the Art and Beyond","volume":"13","author":"Wang","year":"2020","journal-title":"Front. Neurosci."},{"issue":"8","key":"2026081508081860600_cit4","doi-asserted-by":"publisher","first-page":"909","DOI":"10.1517\/14728220903018957","article-title":"Kv1.3 potassium channels as a therapeutic target\nin multiple sclerosis","volume":"13","author":"Rangaraju","year":"2009","journal-title":"Expert Opin. Ther. 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Res.\nRev."},{"issue":"11","key":"2026081508081860600_cit8","doi-asserted-by":"publisher","first-page":"9928","DOI":"10.1074\/jbc.M212868200","article-title":"A novel fluorescent toxin to detect and investigate Kv1.3 channel\nup-regulation in chronically activated T lymphocytes","volume":"278","author":"Beeton","year":"2003","journal-title":"J. Biol. 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Cell Biol."},{"issue":"7","key":"2026081508081860600_cit14","doi-asserted-by":"publisher","first-page":"2379","DOI":"10.1007\/s00216-012-6655-6","article-title":"Fluorescent system based on bacterial expression of hybrid KcsA channels\ndesigned for Kv1.3 ligand screening and study","volume":"405","author":"Kudryashova","year":"2013","journal-title":"Anal. Bioanal. Chem."},{"issue":"1","key":"2026081508081860600_cit15","doi-asserted-by":"publisher","first-page":"33314","DOI":"10.1038\/srep33314","article-title":"Fluorescent\nprotein-scorpion toxin chimera is a convenient\nmolecular tool for studies of potassium channels","volume":"6","author":"Kuzmenkov","year":"2016","journal-title":"Sci. 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Fungi."},{"issue":"19","key":"2026081508081860600_cit23","doi-asserted-by":"publisher","first-page":"4049","DOI":"10.1021\/bi300060n","article-title":"Structure, function,\nand chemical synthesis of Vaejovis mexicanus\npeptide 24: a novel potent blocker of Kv1.3 potassium channels of\nhuman T lymphocytes","volume":"51","author":"Gurrola","year":"2012","journal-title":"Biochemistry"},{"issue":"8","key":"2026081508081860600_cit24","doi-asserted-by":"publisher","first-page":"1753","DOI":"10.1038\/sj.jid.5700329","article-title":"In Vivo Monitoring of Cutaneous Edema\nusing Spectral Imaging in the Visible and Near Infrared","volume":"126","author":"Stamatas","year":"2006","journal-title":"J. Invest. Dermatol."},{"issue":"2\u20133","key":"2026081508081860600_cit25","doi-asserted-by":"publisher","first-page":"90","DOI":"10.1016\/S1011-1344(00)00082-8","article-title":"Measurement of the penetration\ndepths of red and near infrared light\nin human \u201cex vivo\u201d tissues","volume":"57","author":"Stolik","year":"2000","journal-title":"J.\nPhotochem. Photobiol. B"},{"issue":"8","key":"2026081508081860600_cit26","doi-asserted-by":"publisher","first-page":"1909","DOI":"10.1007\/s10103-017-2317-4","article-title":"Effect of wavelength\nand beam width on penetration in light-tissue\ninteraction using computational methods","volume":"32","author":"Ash","year":"2017","journal-title":"Lasers\nMed. 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Technol."},{"issue":"1","key":"2026081508081860600_cit32","doi-asserted-by":"publisher","first-page":"69","DOI":"10.1016\/j.procbio.2011.10.009","article-title":"Identification of cell\nculture conditions to control protein aggregation\nof IgG fusion proteins expressed in Chinese hamster ovary cells","volume":"47","author":"Jing","year":"2012","journal-title":"Process Biochem."},{"issue":"5","key":"2026081508081860600_cit33","doi-asserted-by":"publisher","first-page":"2639","DOI":"10.1074\/jbc.273.5.2639","article-title":"Subunit composition\nof brain voltage-gated potassium channels determined\nby hongotoxin-1, a novel peptide derived from Centruroides limbatus\nvenom","volume":"273","author":"Koschak","year":"1998","journal-title":"J. Biol. Chem."},{"key":"2026081508081860600_cit34","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1007\/978-94-007-6404-0_30","article-title":"Potassium\nChannel Blocking Peptide\nToxins from Scorpion Venom","volume-title":"Scorpion Venoms","author":"Bartok","year":"2015"},{"key":"2026081508081860600_cit35","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.00594","article-title":"Structure Of a Pore-Blocking Toxin\nIn Complex With a Eukaryotic Voltage-Dependent K+ Channel","volume":"2","author":"Banerjee","year":"2013","journal-title":"eLife"},{"issue":"34","key":"2026081508081860600_cit36","doi-asserted-by":"publisher","first-page":"7749","DOI":"10.1021\/bi00149a002","article-title":"Mapping function to\nstructure in a channel-blocking\npeptide: electrostatic mutants of charybdotoxin","volume":"31","author":"Park","year":"1992","journal-title":"Biochemistry"},{"key":"2026081508081860600_cit37","doi-asserted-by":"publisher","first-page":"421564","DOI":"10.1155\/2012\/421564","article-title":"Expression and Role of the Intermediate-Conductance\nCalcium-Activated Potassium Channel KCa3.1 in Glioblastoma","volume":"2012","author":"Catacuzzeno","year":"2012","journal-title":"J. Signal Transduction"},{"issue":"13","key":"2026081508081860600_cit38","doi-asserted-by":"publisher","first-page":"1437","DOI":"10.2174\/092986707780831186","article-title":"Modulators\nof Small- and Intermediate-Conductance Calcium-Activated Potassium\nChannels and their Therapeutic Indications","volume":"14","author":"Heike","year":"2007","journal-title":"Curr. Med. Chem."},{"issue":"1","key":"2026081508081860600_cit39","doi-asserted-by":"publisher","first-page":"41","DOI":"10.3390\/toxins15010041","article-title":"Characterization\nand Chemical Synthesis of Cm39 (\u03b1-KTx 4.8):\na Scorpion Toxin That Inhibits Voltage-Gated K\u00ad(+) Channel K\u00ad(V)\u00ad1.2\nand Small- and Intermediate-Conductance Ca\u00ad(2+)-Activated K\u00ad(+) Channels\nK\u00ad(Ca)\u00ad2.2 and K\u00ad(Ca)\u00ad3.1","volume":"15","author":"Naseem","year":"2023","journal-title":"Toxins"},{"issue":"8","key":"2026081508081860600_cit40","doi-asserted-by":"publisher","DOI":"10.1085\/jgp.202213146","article-title":"Cm28, a scorpion toxin\nhaving a unique primary structure, inhibits KV1.2 and KV1.3 with high\naffinity","volume":"154","author":"Naseem","year":"2022","journal-title":"J. Gen. Physiol."},{"issue":"6","key":"2026081508081860600_cit41","doi-asserted-by":"publisher","first-page":"279","DOI":"10.3390\/toxins17060279","article-title":"Cvill6\nand Cvill7: Potent and Selective Peptide Blockers\nof Kv1.2 Ion Channel Isolated from Mexican Scorpion Centruroides villegasi","volume":"17","author":"Shakeel","year":"2025","journal-title":"Toxins"},{"issue":"11","key":"2026081508081860600_cit42","doi-asserted-by":"publisher","first-page":"2197","DOI":"10.1021\/acs.bioconjchem.2c00436","article-title":"A Fluorescent Peptide\nToxin for Selective Visualization\nof the Voltage-Gated Potassium Channel KV1.3","volume":"33","author":"Wai","year":"2022","journal-title":"Bioconjugate Chem."},{"issue":"1","key":"2026081508081860600_cit43","doi-asserted-by":"publisher","first-page":"116","DOI":"10.1186\/s12974-019-1510-8","article-title":"Temporal profiling of\nKv1.3 channel expression in brain mononuclear\nphagocytes following ischemic stroke","volume":"16","author":"Gao","year":"2019","journal-title":"J. Neuroinflammation"},{"issue":"11","key":"2026081508081860600_cit44","doi-asserted-by":"publisher","first-page":"1703","DOI":"10.1172\/JCI16921","article-title":"The voltage-gated Kv1.3\nK + channel in effector memory T cells as\nnew target for MS","volume":"111","author":"Wulff","year":"2003","journal-title":"J.Clin. Invest."},{"issue":"46","key":"2026081508081860600_cit45","doi-asserted-by":"publisher","first-page":"17414","DOI":"10.1073\/pnas.0605136103","article-title":"Kv1.3 channels are a therapeutic target for\nT cell-mediated autoimmune diseases","volume":"103","author":"Beeton","year":"2006","journal-title":"Proc. Natl.\nAcad. Sci. U. S. A."},{"key":"2026081508081860600_cit46","doi-asserted-by":"publisher","first-page":"714841","DOI":"10.3389\/fphar.2021.714841","article-title":"Kv1.3 Channel Up-Regulation in Peripheral Blood T Lymphocytes of\nPatients With Multiple Sclerosis","volume":"12","author":"Markakis","year":"2021","journal-title":"Front. Pharmacol."},{"key":"2026081508081860600_cit47","doi-asserted-by":"publisher","first-page":"933","DOI":"10.3389\/fonc.2019.00933","article-title":"Voltage-Gated Potassium\nChannel Kv1.3 as a Target in Therapy of Cancer","volume":"9","author":"Teisseyre","year":"2019","journal-title":"Front. Oncol."},{"issue":"3","key":"2026081508081860600_cit48","doi-asserted-by":"publisher","first-page":"171","DOI":"10.1111\/j.1365-2184.1969.tb00229.x","article-title":"The Kinetics\nOf Lymphocyte Recirculation Within The Rat Spleen","volume":"2","author":"Ford","year":"1969","journal-title":"Cell Prolif."},{"issue":"6","key":"2026081508081860600_cit49","doi-asserted-by":"publisher","first-page":"529","DOI":"10.1111\/j.1365-2184.1975.tb01239.x","article-title":"The predominant role\nof the spleen in lymphocyte recirculation.\nI. 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