Oral Presentation: 20 minutes 11th Asia-Pacific Congress of the International Society on Toxinology 2021

A fluorescently-tagged peptide toxin as a tool for KV1.3 visualisation  (#48)

Dorothy C Wai 1 , Sanjeevini Babu Reddiar 1 , Yijun Pan 1 , Gabor Mocsar 2 , Ágota Csóti 2 , Gyorgy Panyi 2 , Joseph Nicolazzo 1 , Ray Norton 1
  1. Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, VIC, Australia
  2. Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary

The voltage-gated potassium channel KV1.3 plays an important role in T cell and microglia activation [1, 2]. Homotetrameric KV1.3 channels are specifically upregulated in effector memory T-cells, which have been implicated in autoimmune diseases including rheumatoid arthritis, psoriasis, multiple sclerosis and type I diabetes [3]. Microglia, which also express KV1.5, upregulate KV1.3 in neuroinflammatory conditions such as Alzheimer’s and Parkinson’s disease [4]. It is of interest to be able to specifically identify and visualise homotetrameric KV1.3 channels, which have distinct functional properties from other KV1 channels and KV1.3 heterotetramers [5]. Fluorescent conjugates of antibodies or small molecules are unable to distinguish homotetrameric KV1.3 channels from heterotetrameric KV1.3/KV1.x channels. In contrast, a number of animal-derived peptide toxins that bind the extracellular vestibule of the channel exhibit selectivity for KV1.3 homotetramers [6].

HsTX1[R14A] is an analogue of a 34-residue peptide toxin from the scorpion Heterometrus spinifer that binds KV1.3 with an IC50 of 45 pM and displays a 2000-fold selectivity for KV1.3 over KV1.1 [7]. We have synthesised a fluorescent analogue of HsTX1[R14A] by N-terminal conjugation of a Cy5 tag. Electrophysiology assays show that Cy5-HsTX1[R14A] retains nanomolar activity against KV1.3 and selectivity over KV1.5. Live-cell imaging of CHO cells expressing GFP-KV1.3 shows colocalization of Cy5-HsTX1[R14A] and KV1.3 fluorescence signals at the cell membrane. Cy5-HsTX1[R14A] is also able to detect KV1.3 upregulation at physiologically relevant expression levels in lipopolysaccharide-stimulated mouse microglia. Furthermore, the tissue-penetrating far-red emission profile of Cy5 affords potential for visualising peptide and KV1.3 biodistribution in vivo, as illustrated by our preliminary studies in mice. These results highlight the utility of Cy5-HsTX1[R14A] as a KV1.3 probe, which will have broad applicability in fundamental investigations of KV1.3 trafficking and KV1 channel composition, as well as in the validation of novel disease indications where KV1.3 inhibition may be of therapeutic value.

 

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