N- or C-terminal Position of the Fluorescent Protein mKate2 in the mKate2-KCa3.1 Chimera Influences Membrane Expression of the Channel

The intermediate-conductance calcium-activated potassium channel KCa3.1 promotes calcium-dependent 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.