N- or C-terminal position of the fluorescent protein mKate2 in the mKate2-KCa3.1 chimera influences membrane expression of the channel
https://doi.org/10.55959/MSU0137-0952-16-80-3S-7
Abstract
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.
Supporting agencies: The research was supported by the Russian Science Foundation, project no. 22-1400406P.
About the Authors
V. N. Korabeynikova
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences; Faculty of Biology, Lomonosov Moscow State University
Russian Federation
Russian Federation
16/10 Miklukho-Maklaya Str., Moscow, 117997
1–12 Leninskie gory, Moscow, 119234
A. V. Feofanov
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences; Faculty of Biology, Lomonosov Moscow State University
Russian Federation
Russian Federation
16/10 Miklukho-Maklaya Str., Moscow, 117997
1–12 Leninskie gory, Moscow, 119234
O. V. Nekrasova
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
Russian Federation
Russian Federation
16/10 Miklukho-Maklaya Str., Moscow, 117997
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Review
For citations:
Korabeynikova V.N., Feofanov A.V., Nekrasova O.V. N- or C-terminal position of the fluorescent protein mKate2 in the mKate2-KCa3.1 chimera influences membrane expression of the channel. Vestnik Moskovskogo universiteta. Seriya 16. Biologiya. 2025;80(3):46–51. (In Russ.) https://doi.org/10.55959/MSU0137-0952-16-80-3S-7
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