vestnik-bio-msuВестник Московского университета. Серия 16. БиологияVestnik Moskovskogo universiteta. Seriya 16. Biologiya0137-0952Lomonosov Moscow State University, School of Biology10.55959/MSU0137-0952-16-80-3S-7vestnik-bio-msu-1540Research ArticleОригинальные исследованияN- или C-концевое положение флуоресцентного белка mKate2 в составе химеры mKate2-KCa3.1 влияет на мембранную экспрессию каналаN- or C-terminal position of the fluorescent protein mKate2 in the mKate2-KCa3.1 chimera influences membrane expression of the channelКорабейниковаВ Н.KorabeynikovaV. N.

Корабейникова Варвара Николаевна – инженер лаборатории оптической микроскопии и спектроскопии биомолекул 

117997, г. Москва, ул. Миклухо-Маклая, д. 16/10 

119234, г. Москва, Ленинские горы, д. 1, стр. 12 

 Тел.: 8-495-335-01-00 

 16/10 Miklukho-Maklaya Str., Moscow, 117997 

 1–12 Leninskie gory, Moscow, 119234 

var.kora.3@gmail.comhttps://orcid.org/0000-0002-1596-9506ФеофановА. В.FeofanovA. V.

Феофанов Алексей Валерьевич – докт. биол. наук., гл. науч. сотр., зав. лабораторией оптической микроскопии и спектроскопии биомолекул ИБХ РАН; проф. кафедры биоинженерии биологического факультета МГУ 

117997, г. Москва, ул. Миклухо-Маклая, д. 16/10 

119234, г. Москва, Ленинские горы, д. 1, стр. 12 

 Тел.: 8-495-335-01-00 

 16/10 Miklukho-Maklaya Str., Moscow, 117997 

 1–12 Leninskie gory, Moscow, 119234 

avfeofanov@yandex.ruhttps://orcid.org/0000-0002-7216-1618НекрасоваО. В.NekrasovaO. V.

Некрасова Оксана Васильевна – канд. биол. наук, ст. науч. сотр. лаборатории инженерии белка 

117997, г. Москва, ул. Миклухо-Маклая, д. 16/10 

Тел.: 8-495-335-01-00 

 16/10 Miklukho-Maklaya Str., Moscow, 117997 

okatja@yandex.ruИнститут биоорганической химии имени академиков М.М. Шемякина и Ю.А. Овчинникова, Российская академия наук; Биологический факультет, Московский государственный университет имени М.В. ЛомоносоваРоссияShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences; Faculty of Biology, Lomonosov Moscow State UniversityRussian FederationИнститут биоорганической химии имени академиков М.М. Шемякина и Ю.А. Овчинникова, Российская академия наукРоссияShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of SciencesRussian Federation202510112025803S46–51Copyright © Корабейникова В.Н., Феофанов А.В., Некрасова О.В., 20252025Корабейникова В.Н., Феофанов А.В., Некрасова О.В.Korabeynikova V.N., Feofanov A.V., Nekrasova O.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestnik-bio-msu.elpub.ru/jour/article/view/1540

Активируемый кальцием калиевый канал промежуточной проводимости KCa3.1 способствует Ca2+-зависимой гиперполяризации клеточной мембраны, а нарушения его работы наблюдаются при аутоиммунных и онкологических заболеваниях. Для изучения этого канала и его пептидных блокаторов с использованием флуоресцентного анализа были сконструированы плазмиды, кодирующие α-субъединицу KCa3.1, слитую с флуоресцентным белком mKate2 на N- или C-конце, а также получен флуоресцентный лиганд ChTxGFP, объединяющий в себе пептидный блокатор харибдотоксин и зеленый флуоресцентный белок. Установлено, что mKate2 на N-конце α-субъединицы блокирует перенос канала в плазматическую мембрану клеток Neuro-2а, тогда как mKate2 на ее С-конце не препятствует эффективному накоплению канала в плазматической мембране и формированию его правильной тетрамерной структуры, способной связывать пептидные блокаторы. Лиганд ChTx-GFP связывается на мембране с каналом KCa3.1 при концентрации 20 нМ и может быть использован для флуоресцентного имиджинга этих каналов в клетках млекопитающих.

The intermediate-conductance calcium-activated potassium channel KCa3.1 promotes calciumdependent hyperpolarization of the cell membrane. Its malfunction has been observed in autoimmune and oncological diseases. To study this channel and its peptide blockers using fluorescence analysis, plasmids encoding the α-subunit KCa3.1 fused with the fluorescent protein mKate2 at the N- or C-terminus were constructed, and the fluorescent ligand ChTx-GFP was obtained, which is a combination of the peptide blocker charybdotoxin and the green fluorescent protein. It was found that mKate2 at the N-terminus of the α-subunit blocks the transport of the channel into the plasma membrane of Neuro-2a cells, while mKate2 at its C-terminus does not interfere with the efficient accumulation of the channel in the plasma membrane and the formation of a regular tetrameric structure capable of binding peptide blockers. The ligand ChTx-GFP binds to the KCa3.1 channel on the membrane at a concentration of 20 nM and can be used for fluorescent imaging of these channels in mammalian cells.

калиевые каналыканал KCa3.1пептидные блокаторыфлуоресценцияконфокальная микроскопияpotassium channelschannel KCa3.1peptide blockersfluorescenceconfocal microscopyРабота выполнена при финансовой поддержке Российского научного фонда (проект №22- 1400406П).The research was supported by the Russian Science Foundation, project no. 22-1400406P.ReferencesOrfali R., Albanyan N. Ca2+-sensitive potassium channels. Molecules. 2023; 28(2):885.Orfali R., Albanyan N. Ca2+-sensitive potassium channels. Molecules. 2023; 28(2):885.Ghanshani S., Wulff H., Miller M.J., Rohm H., Neben A., Gutman G.A., Cahalan M.D., Chandy K.G. Up-regulation of the IKCa1 potassium channel during T-cell activation: molecular mechanism and functional consequences. J. Biol. Chem. 2000;275(47):37137–37149.Ghanshani S., Wulff H., Miller M.J., Rohm H., Neben A., Gutman G.A., Cahalan M.D., Chandy K.G. Up-regulation of the IKCa1 potassium channel during T-cell activation: molecular mechanism and functional consequences. J. Biol. Chem. 2000;275(47):37137–37149.Calderón Artavia C.G., Arvelo Álvarez F.A. Kca3.1- related cellular signalling involved in cancer proliferation. Cell. Physiol. Biochem. Int. J. Exp. Cell. Physiol. Biochem. Pharmacol. 2024;58(2):107–127.Calderón Artavia C.G., Arvelo Álvarez F.A. Kca3.1- related cellular signalling involved in cancer proliferation. Cell. Physiol. Biochem. Int. J. Exp. Cell. Physiol. Biochem. Pharmacol. 2024;58(2):107–127.Cahalan M.D., Chandy K.G. The functional network of ion channels in T lymphocytes. Immunol. Rev. 2009;231(1):59–87.Cahalan M.D., Chandy K.G. The functional network of ion channels in T lymphocytes. Immunol. Rev. 2009;231(1):59–87.Deutsch C., Chen L.Q. Heterologous expression of specific K+ channels in T lymphocytes: functional consequences for volume regulation. Proc. Natl. Acad. Sci. U.S.A. 1993;90(21):10036–10040.Deutsch C., Chen L.Q. Heterologous expression of specific K+ channels in T lymphocytes: functional consequences for volume regulation. Proc. Natl. Acad. Sci. U.S.A. 1993;90(21):10036–10040.Cruse G., Duffy S.M., Brightling C.E., Bradding P. Functional KCa3.1 K+ channels are required for human lung mast cell migration. Thorax. 2006;61(10):880–885.Cruse G., Duffy S.M., Brightling C.E., Bradding P. Functional KCa3.1 K+ channels are required for human lung mast cell migration. Thorax. 2006;61(10):880–885.Szabò I., Zoratti M., Gulbins E. Contribution of voltage-gated potassium channels to the regulation of apoptosis. FEBS Lett. 2010;584(10):2049–2056. 8. Chou C.C., Lunn C.A., Murgolo N.J. KCa3.1: target and marker for cancer, autoimmune disorder and vascular inflammation? Expert Rev. Mol. Diagn. 2008;8(2):179–187.Szabò I., Zoratti M., Gulbins E. Contribution of voltage-gated potassium channels to the regulation of apoptosis. FEBS Lett. 2010;584(10):2049–2056. 8. Chou C.C., Lunn C.A., Murgolo N.J. KCa3.1: target and marker for cancer, autoimmune disorder and vascular inflammation? Expert Rev. Mol. Diagn. 2008;8(2):179–187.Brown B.M., Shim H., Christophersen P., Wulff H. Pharmacology of small-and intermediate-conductance calcium-activated potassium channels. Annu. Rev. Pharmacol. Toxicol. 2020;60(1):219–240.Brown B.M., Shim H., Christophersen P., Wulff H. Pharmacology of small-and intermediate-conductance calcium-activated potassium channels. Annu. Rev. Pharmacol. Toxicol. 2020;60(1):219–240.Visan V., Sabatier J.M., Grissmer S. Block of maurotoxin and charybdotoxin on human intermediate-conductance calcium-activated potassium channels (hIKCa1). Toxicon. 2004;43(8):973–980.Visan V., Sabatier J.M., Grissmer S. Block of maurotoxin and charybdotoxin on human intermediate-conductance calcium-activated potassium channels (hIKCa1). Toxicon. 2004;43(8):973–980.Pimentel C., M’Barek S., Visan V., Grissmer S., Sampieri F., Sabatier J.M., Darbon H., Fajloun Z. Chemical synthesis and 1H-NMR 3D structure determination of AgTx2-MTX chimera, a new potential blocker for Kv1.2 channel, derived from MTX and AgTx2 scorpion toxins. Protein Sci. 2008;17(1):107–118.Pimentel C., M’Barek S., Visan V., Grissmer S., Sampieri F., Sabatier J.M., Darbon H., Fajloun Z. Chemical synthesis and 1H-NMR 3D structure determination of AgTx2-MTX chimera, a new potential blocker for Kv1.2 channel, derived from MTX and AgTx2 scorpion toxins. Protein Sci. 2008;17(1):107–118.M’Barek S., Chagot B., Andreotti N., Visan V., Mansuelle P., Grissmer S., Marrakchi M., El Ayeb M., Sampieri F., Darbon H., Fajloun Z., De Waard M., Sabatier J.M. Increasing the molecular contacts between maurotoxin and Kv1.2 channel augments ligand affinity. Proteins Struct. Funct. Bioinforma. 2005;60(3):401–411.M’Barek S., Chagot B., Andreotti N., Visan V., Mansuelle P., Grissmer S., Marrakchi M., El Ayeb M., Sampieri F., Darbon H., Fajloun Z., De Waard M., Sabatier J.M. Increasing the molecular contacts between maurotoxin and Kv1.2 channel augments ligand affinity. Proteins Struct. Funct. Bioinforma. 2005;60(3):401–411.Rauer H., Lanigan M.D., Pennington M.W., Aiyar J., Ghanshani S., Cahalan M.D., Norton R.S., Chandy K.G. Structure-guided transformation of charybdotoxin yields an analog that selectively targets Ca2+-activated over voltage-gated K+ channels. J. Biol. Chem. 2000;275(2):1201–1208.Rauer H., Lanigan M.D., Pennington M.W., Aiyar J., Ghanshani S., Cahalan M.D., Norton R.S., Chandy K.G. Structure-guided transformation of charybdotoxin yields an analog that selectively targets Ca2+-activated over voltage-gated K+ channels. J. Biol. Chem. 2000;275(2):1201–1208.Bal N.V., Oblasov I., Ierusalimsky V.N., Shvadchenko A.M., Fortygina P., Idzhilova O.S., Borodinova A.A., Balaban P.M., Feofanov A.V., Nekrasova O.V., Nikitin E.S. Potassium KCa3.1 channel overexpression deteriorates functionality and availability of channels at the outer cellular membrane. Sci. Rep. 2025;15(1):4928.Bal N.V., Oblasov I., Ierusalimsky V.N., Shvadchenko A.M., Fortygina P., Idzhilova O.S., Borodinova A.A., Balaban P.M., Feofanov A.V., Nekrasova O.V., Nikitin E.S. Potassium KCa3.1 channel overexpression deteriorates functionality and availability of channels at the outer cellular membrane. Sci. Rep. 2025;15(1):4928.Primak A.L., Orlov N.A., Peigneur S., Tytgat J., Ignatova A.A., Denisova K.R., Yakimov S.A., Kirpichnikov M.P., Nekrasova O.V., Feofanov A.V. AgTx2-GFP, fluorescent blocker targeting pharmacologically important Kv1.x (x = 1, 3, 6) channels. Toxins. 2023;15(3):229.Primak A.L., Orlov N.A., Peigneur S., Tytgat J., Ignatova A.A., Denisova K.R., Yakimov S.A., Kirpichnikov M.P., Nekrasova O.V., Feofanov A.V. AgTx2-GFP, fluorescent blocker targeting pharmacologically important Kv1.x (x = 1, 3, 6) channels. Toxins. 2023;15(3):229.Ignatova A.A., Kryukova E.V., Novoseletsky V.N., Kazakov O.V., Orlov N.A., Korabeynikova V.N., Larina M.V., Fradkov A.F., Yakimov S.A., Kirpichnikov M.P., Feofanov A.V., Nekrasova O.V. New high-affinity peptide ligands for Kv1.2 channel: Selective blockers and fluorescent probes. Cells. 2024;13(24):2096.Ignatova A.A., Kryukova E.V., Novoseletsky V.N., Kazakov O.V., Orlov N.A., Korabeynikova V.N., Larina M.V., Fradkov A.F., Yakimov S.A., Kirpichnikov M.P., Feofanov A.V., Nekrasova O.V. New high-affinity peptide ligands for Kv1.2 channel: Selective blockers and fluorescent probes. Cells. 2024;13(24):2096.Orlov N.A., Kryukova E.V., Efremenko A.V., Yakimov S.A., Toporova V.A., Kirpichnikov M.P., Nekrasova O.V., Feofanov A.V. Interactions of the Kv1. 1 channel with peptide pore blockers: a fluorescent analysis on mammalian cells. Membranes. 2023;13(7):645.Orlov N.A., Kryukova E.V., Efremenko A.V., Yakimov S.A., Toporova V.A., Kirpichnikov M.P., Nekrasova O.V., Feofanov A.V. Interactions of the Kv1. 1 channel with peptide pore blockers: a fluorescent analysis on mammalian cells. Membranes. 2023;13(7):645.Porta E.A. Pigments in aging: An overview. Ann. N. Y. Acad. Sci. 2002;959(1):57–65.Porta E.A. Pigments in aging: An overview. Ann. N. Y. Acad. Sci. 2002;959(1):57–65.Nekrasova O.V., Volyntseva A.D., Kudryashova K.S., Novoseletsky V.N., Lyapina E.A., Illarionova A.V., Yakimov S.A., Korolkova Yu.V., Shaitan K.V., Kirpichnikov M.P., Feofanov A.V. Complexes of peptide blockers with Kv1.6 pore domain: Molecular modeling and studies with KcsA-Kv1.6 channel. J. Neuroimmune Pharmacol. 2017;12(2):260–276.Nekrasova O.V., Volyntseva A.D., Kudryashova K.S., Novoseletsky V.N., Lyapina E.A., Illarionova A.V., Yakimov S.A., Korolkova Yu.V., Shaitan K.V., Kirpichnikov M.P., Feofanov A.V. Complexes of peptide blockers with Kv1.6 pore domain: Molecular modeling and studies with KcsA-Kv1.6 channel. J. Neuroimmune Pharmacol. 2017;12(2):260–276.Denisova K.R., Orlov N.A., Yakimov S.A., Kirpichnikov M.P., Feofanov A.V., Nekrasova O.V. Atto488-agitoxin 2—a fluorescent ligand with increased selectivity for Kv1.3 channel binding site. Bioengineering. 2022;9(7):295.Denisova K.R., Orlov N.A., Yakimov S.A., Kirpichnikov M.P., Feofanov A.V., Nekrasova O.V. Atto488-agitoxin 2—a fluorescent ligand with increased selectivity for Kv1.3 channel binding site. Bioengineering. 2022;9(7):295.Jones H.M., Hamilton K.L., Papworth G.D., Syme C.A., Watkins S.C., Bradbury N.A., Devor D.C. Roleof the NH2 terminus in the assembly and trafficking of the intermediate conductance Ca2+-activated K+ channel hIK1. J. Biol. Chem. 2004;279(15):15531–15540.Jones H.M., Hamilton K.L., Papworth G.D., Syme C.A., Watkins S.C., Bradbury N.A., Devor D.C. Roleof the NH2 terminus in the assembly and trafficking of the intermediate conductance Ca2+-activated K+ channel hIK1. J. Biol. Chem. 2004;279(15):15531–15540.Lee C.H., MacKinnon R. Activation mechanism of a human SK-calmodulin channel complex elucidated by cryo-EM structures. Science. 2018;360(6388):508–513.Lee C.H., MacKinnon R. Activation mechanism of a human SK-calmodulin channel complex elucidated by cryo-EM structures. Science. 2018;360(6388):508–513.Balut C.M., Hamilton K.L., Devor D.C. Trafficking of intermediate (KCa3.1) and small (KCa2.x) conductance, Ca2+-activated K+ channels: a novel target for medicinal chemistry efforts? ChemMedChem. 2012;7(10):1741–1755.Balut C.M., Hamilton K.L., Devor D.C. Trafficking of intermediate (KCa3.1) and small (KCa2.x) conductance, Ca2+-activated K+ channels: a novel target for medicinal chemistry efforts? ChemMedChem. 2012;7(10):1741–1755.Jiménez-Pérez L., Cidad P., Álvarez-Miguel I., Santos-Hipólito A., Torres-Merino R., Alonso E., de la Fuente M.Á., López-López J.R., Pérez-García M.T. Molecular determinants of Kv1.3 potassium channels-induced proliferation. J. Biol. Chem. 2016;291(7):3569–3580.Jiménez-Pérez L., Cidad P., Álvarez-Miguel I., Santos-Hipólito A., Torres-Merino R., Alonso E., de la Fuente M.Á., López-López J.R., Pérez-García M.T. Molecular determinants of Kv1.3 potassium channels-induced proliferation. J. Biol. Chem. 2016;291(7):3569–3580.Spear J.M., Koborssy D.A., Schwartz A.B., Johnson A.J., Audhya A., Fadool D.A., Stagg S.M. Kv1.3 contains an alternative C-terminal ER exit motif and is recruited into COPII vesicles by Sec24a. BMC Biochem. 2015;16(1):16.Spear J.M., Koborssy D.A., Schwartz A.B., Johnson A.J., Audhya A., Fadool D.A., Stagg S.M. Kv1.3 contains an alternative C-terminal ER exit motif and is recruited into COPII vesicles by Sec24a. BMC Biochem. 2015;16(1):16.Vicente R., Escalada A., Villalonga N., Texidó L., Roura-Ferrer M., Martín-Satué M., López-Iglesias C., Soler C., Solsona C., Tamkun M.M., Felipe A. Association of Kv1.5 and Kv1.3 Contributes to the major voltagedependent K+ channel in macrophages. J. Biol. Chem. 2006;281(49):37675–37685.Vicente R., Escalada A., Villalonga N., Texidó L., Roura-Ferrer M., Martín-Satué M., López-Iglesias C., Soler C., Solsona C., Tamkun M.M., Felipe A. Association of Kv1.5 and Kv1.3 Contributes to the major voltagedependent K+ channel in macrophages. J. Biol. Chem. 2006;281(49):37675–37685.

The authors declare that there are no conflicts of interest present.