A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel K_V1.3

Abstract

Upregulation of the voltage-gated potassium channel K V 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 V 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 Heterometrus spinifer, binds K V 1.3 with high affinity (IC 50 of 45 pM) and selectivity (2000-fold for K V 1.3 over K V 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 V 1.3 (IC 50 ∼ 0.9 nM) and selectivity over a range of other potassium channels (K.1 and K Ca 3.1), as well as selectivity against heteromeric channels assembled from K V 1.3/K V 1.5 tandem dimers. Live imaging of CHO cells expressing green fluorescent protein-tagged K V 1.3 shows co-localization of Cy5−HsTX1[R14A] and K V 1.3 fluorescence signals at the cell membrane. Moreover, flow cytometry demonstrated that Cy5−HsTX1[R14A] can detect K V 1.3-expressing CHO cells. Stimulation of mouse microglia by lipopolysaccharide, which enhances membrane expression of K V 1.3, was associated with increased staining by Cy5−HsTX1[R14A], demonstrating that it can be used to identify K V 1.3 in disease-relevant models of inflammation. Furthermore, the biodistribution of Cy5−HsTX1[R14A] could be monitored using ex vivo 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 V 1.3, with broad applicability in fundamental investigations of K V 1.3 biology, and the validation of novel disease indications where K V 1.3 inhibition may be of therapeutic value.

Neurotoxin-Derived Optical Probes for Biological and Medical Imaging

“…To demonstrate the specificity and high quantum efficacy of QDs-MgTx, it was applied to HEK293 cells transfected with GFP tagged K V 1.3 construct, and showed a strong co-localisation of QDs with GFP, yielding a more stable and stronger QD signal [ 57 ]. A very recent study showed that conjugation of the scorpion Heterometrus spinifer -derived HsTX1 analogue HsTX1 [R14A] with Cy5 allowed the visualisation of GFP-K V 1.3 channels in CHO cells [ 58 ]. Cy5-HsTX1 enabled also imaging of Kv1.3 in BV-2 microglia cells of C57BL/6 mice treated with lipopolysaccharides (LPS), known to upregulate K V 1.3 subunit.…”

“…The presence of fluorescence signals in the kidney, intestine and liver of C57BL/6 mice injected with Cy5-HsTX1 [R14A] implies its potential usefulness for biodistribution studies. Authors suggest that Cy5-HsTX1 [R14A] is a useful probe to determine the location and distribution of K V 1.3 channels under physiological as well as autoimmune and neuroinflammatory conditions, associated with the upregulation of K V 1.3 channels [ 58 ].…”

AgTx2-GFP, Fluorescent Blocker Targeting Pharmacologically Important Kv1.x (x = 1, 3, 6) Channels

“…In many cases, the labeling strategy includes the construction of a mutated peptide (by inserting Tyr or Cys residue) with retained biological activity, followed by the iodination of Tyr with 125 I [ 36 , 37 ], and the labeling of Cys with [ 3 H]-N-ethylmaleimide [ 38 ] or with thiol-reactive fluorescent dyes [ 39 , 40 ]. Other approaches are based on the labeling of the N-terminal amino group with an organic fluorophore during solid-phase synthesis of the peptide [ 41 , 42 , 43 ] or the “blind” labeling of one of several Lys residues, which are present in the peptide, with an amino-reactive organic fluorophore [ 44 ]. In the latter case, the separation of labeled derivatives and the selection of the most active ones followed by the determination of the modified Lys residue are required.…”

Synthesis, Activity, and Application of Fluorescent Analogs of [D1G, Δ14Q]LvIC Targeting α6β4 Nicotinic Acetylcholine Receptor

“…19,28,29 Scorpion toxins targeting voltage-gated potassium ion (K V ) channels could also be linked to different fluorescent molecules, such as HsTX1[R14A], OSK1, and AgTx2, serving as pharmacological tools for studying K V channels. 30,31 In addition, a labeled toxin for imaging the rat TRPV1 ion channel could be obtained by connecting an agonist double-knot toxin (DkTx) with fluorescent molecules. 32 Together, these results indicate that peptide toxins with high activity and selectivity for specific subtypes of ion channels can serve as ideal pharmacological tools for visualizing and imaging ion channels when linked to various fluorescent molecules.…”

“…For example, the fluorescent molecule 5-carboxytetramethylrhodamine succinimidyl ester (5-TAMRA-SE) could react with the N-terminus of α-CTx LtIA targeting α3β2 nAChR to obtain a novel fluorescent analog of LtIA . Other α-CTxs, such as MII and TxID (targeting α3β2 and α3β4 nAChRs, respectively), could also connect with different fluorescent molecules, providing pharmacological tools for exploring the functional neuroanatomy of various subtypes of nAChRs. ,, Scorpion toxins targeting voltage-gated potassium ion (K V ) channels could also be linked to different fluorescent molecules, such as HsTX1­[R14A], OSK1, and AgTx2, serving as pharmacological tools for studying K V channels. , In addition, a labeled toxin for imaging the rat TRPV1 ion channel could be obtained by connecting an agonist double-knot toxin (DkTx) with fluorescent molecules . Together, these results indicate that peptide toxins with high activity and selectivity for specific subtypes of ion channels can serve as ideal pharmacological tools for visualizing and imaging ion channels when linked to various fluorescent molecules.…”