| Autor: |
Wai, Dorothy C. C., Naseem, Muhammad Umair, Mocsár, Gábor, Babu Reddiar, Sanjeevini, Pan, Yijun, Csoti, Agota, Hajdu, Peter, Nowell, Cameron, Nicolazzo, Joseph A., Panyi, Gyorgy, Norton, Raymond S. |
| Zdroj: |
Bioconjugate Chemistry; 20220101, Issue: Preprints |
| Abstrakt: |
Upregulation of the voltage-gated potassium channel KV1.3 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 KV1.3 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 Heterometrus spinifer, binds KV1.3 with high affinity (IC50of 45 pM) and selectivity (2000-fold for KV1.3 over KV1.1). 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 KV1.3 (IC50∼ 0.9 nM) and selectivity over a range of other potassium channels (KV1.2, KV1.4, KV1.5, KV1.6, KCa1.1 and KCa3.1), as well as selectivity against heteromeric channels assembled from KV1.3/KV1.5 tandem dimers. Live imaging of CHO cells expressing green fluorescent protein-tagged KV1.3 shows co-localization of Cy5–HsTX1[R14A] and KV1.3 fluorescence signals at the cell membrane. Moreover, flow cytometry demonstrated that Cy5–HsTX1[R14A] can detect KV1.3-expressing CHO cells. Stimulation of mouse microglia by lipopolysaccharide, which enhances membrane expression of KV1.3, was associated with increased staining by Cy5–HsTX1[R14A], demonstrating that it can be used to identify KV1.3 in disease-relevant models of inflammation. Furthermore, the biodistribution of Cy5–HsTX1[R14A] could be monitored using ex vivofluorescence imaging of organs in mice dosed subcutaneously with the peptide. These results illustrate the utility of Cy5–HsTX1[R14A] as a tool for visualizing KV1.3, with broad applicability in fundamental investigations of KV1.3 biology, and the validation of novel disease indications where KV1.3 inhibition may be of therapeutic value. |
| Databáze: |
Supplemental Index |
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