Voltage-gated potassium channels play a key role in T-cell activation [1]. Homotetrameric K_V1.3 channels are specifically upregulated in effector memory T-cells (T_EM), which have been implicated in autoimmune diseases including rheumatoid arthritis, psoriasis, multiple sclerosis and type I diabetes [2]. It is therefore of interest to be able to identify such K_V1.3-expressing cells. However, fluorescent conjugates of antibodies or small molecules may not be able to distinguish homotetrameric K_V1.3 channels from heterotetrameric K_V1.3/K_V1.x channels. In contrast, a number of animal-derived peptide toxins that bind the extracellular vestibule of the channel exhibit selectivity for K_V1.3 homotetramers [3]. One example is HsTX1[R14A], an analogue of a 34-residue peptide toxin from the scorpion Heterometrus spinifer that binds K_V1.3 with an IC₅₀ of 45 pM and displays a 2000-fold selectivity for K_V1.3 over K_V1.1 [4].

     Labelling of peptide toxins with small-molecule fluorophores is potentially costly and synthetically challenging as residues commonly used for fluorophore attachment may also be critical for toxin folding and function [5]. Earlier work has demonstrated the feasibility of tagging peptide toxins with fluorescent proteins by recombinant expression [6]. A possible limitation is that many peptide toxins require oxidising conditions to form native disulfide bonds. To address this issue, we have developed a construct comprising HsTX1[R14A] and an N-terminal GFP variant [7] containing Cys-to-Ser mutations compatible with an oxidising expression environment. The fusion protein was successfully produced by periplasmic expression in E. coli, purified by nickel affinity and size-exclusion chromatography, and assessed by UV-Vis spectroscopy and ¹H 1D NMR. We are currently optimising the length and composition of the linker between GFP and HsTX1[R14A], with variants to be evaluated for K_V1.3 inhibition using electrophysiology assays. Once validated, GFP-HsTX1[R14A] would be an accessible and cost-effective tool for studying K_V1.3-expressing cells in applications such as confocal imaging and fluorescence-activated cell sorting.