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Upregulation of the voltage-gated potassium channel K
1.3 is implicated in a range of autoimmune and neuroinflammatory diseases, including rheumatoid arthritis, psoriasis, multiple sclerosis, and type I diabetes. Understanding the expression, localization, and trafficking of K
1.3 in normal and disease states is key to developing targeted immunomodulatory therapies. HsTX1[R14A], an analogue of a 34-residue peptide toxin from the scorpion
, binds K
1.3 with high affinity (IC
of 45 pM) and selectivity (2000-fold for K
1.3 over K
1.1). We have synthesized a fluorescent analogue of HsTX1[R14A] by N-terminal conjugation of a Cy5 tag. Electrophysiology assays show that Cy5-HsTX1[R14A] retains activity against K
1.3 (IC
∼ 0.9 nM) and selectivity over a range of other potassium channels (K
1.2, K
1.4, K
1.5, K
1.6, K
1.1 and K
3.1), as well as selectivity against heteromeric channels assembled from K
1.3/K
1.5 tandem dimers. Live imaging of CHO cells expressing green fluorescent protein-tagged K
1.3 shows co-localization of Cy5-HsTX1[R14A] and K
1.3 fluorescence signals at the cell membrane. Moreover, flow cytometry demonstrated that Cy5-HsTX1[R14A] can detect K
1.3-expressing CHO cells. Stimulation of mouse microglia by lipopolysaccharide, which enhances membrane expression of K
1.3, was associated with increased staining by Cy5-HsTX1[R14A], demonstrating that it can be used to identify K
1.3 in disease-relevant models of inflammation. Furthermore, the biodistribution of Cy5-HsTX1[R14A] could be monitored using
fluorescence imaging of organs in mice dosed subcutaneously with the peptide. These results illustrate the utility of Cy5-HsTX1[R14A] as a tool for visualizing K
1.3, with broad applicability in fundamental investigations of K
1.3 biology, and the validation of novel disease indications where K
1.3 inhibition may be of therapeutic value.