Kuzmenkov AI et al. (2016), Fluorescent protein-scorpion toxin chimera is a...

XB-ART-55039

Sci Rep 2016 Sep 21;6:33314. doi: 10.1038/srep33314.

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Fluorescent protein-scorpion toxin chimera is a convenient molecular tool for studies of potassium channels.

Kuzmenkov AI , Nekrasova OV , Kudryashova KS , Peigneur S , Tytgat J , Stepanov AV , Kirpichnikov MP , Grishin EV , Feofanov AV , Vassilevski AA .

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Ion channels play a central role in a host of physiological and pathological processes and are the second largest target for existing drugs. There is an increasing need for reliable tools to detect and visualize particular ion channels, but existing solutions suffer from a number of limitations such as high price, poor specificity, and complicated protocols. As an alternative, we produced recombinant chimeric constructs (FP-Tx) consisting of fluorescent proteins (FP) fused with potassium channel toxins from scorpion venom (Tx). In particular, we used two FP, eGFP and TagRFP, and two Tx, OSK1 and AgTx2, to create eGFP-OSK1 and RFP-AgTx2. We show that these chimeras largely retain the high affinity of natural toxins and display selectivity to particular ion channel subtypes. FP-Tx are displaced by other potassium channel blockers and can be used as an imaging tool in ion channel ligand screening setups. We believe FP-Tx chimeras represent a new efficient molecular tool for neurobiology.

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	Figure 1. Molecular model of complex formed between eGFP-OSK1 and KV.FP-Tx modules are colored: eGFP is in green, and OSK1 is in blue; flexible N-terminus and linker are in gray. The functionally crucial lysine residue of Tx is shown in red pointing to the channel selectivity filter. The model was built based on homology with the known structures of eGFP (PDB ID: 2Y0G), OSK1 (1SCO), and complex of the KV1.2-KV2.1 paddle chimera with charybdotoxin (4JTA).
	Figure 2. FP-Tx fluorescence and binding to KcsA-KV hybrid channels.(A,D) Fluorescence excitation (dashed lines) and emission (solid lines) spectra of eGFP-OSK1 (A) and RFP-AgTx2 (D). Insets show excitation and emission spectra of separate eGFP and TagRFP. (B,E) Relative binding of eGFP-OSK1 (20 nM) and eGFP (20 nM) (B), and RFP-AgTx2 (10 nM) and RFP (10 nM) (E) to KcsA-KV1.3, KcsA-KV1.1, and KcsA-bearing spheroplasts and to spheroplasts without recombinant channels. (C,F) Typical confocal fluorescence images of KcsA-KV1.3-bearing spheroplasts stained with eGFP-OSK1 (C) and KcsA-KV1.1-bearing spheroplasts stained with RFP-AgTx2 (F). The length of the scale bars is 4 μm for both images.
	Figure 3. FP-Tx activity studied by electrophysiology.Shown are representative traces of currents through KV1.1-1.6 channels in control and after application of 1 μM OSK1 (A), and 1 μM eGFP-OSK1 (B), or 0.5 μM AgTx2 (C), and 0.5 μM RFP-AgTx2 (D) (indicated with asterisks).
	Figure 4. Concentration-response curves for FP-Tx on KV1 channels.Comparison between OSK1 and eGFP-OSK1 (A), or AgTx2 and RFP-AgTx2 (B).
	Figure 5. Binding of FP-Tx to KcsA-KV1-bearing spheroplasts and their displacement by different ligands.(A,D) Saturation curves of eGFP-OSK1 and RFP-AgTx2 binding to KcsA-KV1.1 or KcsA-KV1.3-bearing spheroplasts. (B,C) Competition between eGFP-OSK1 and different ligands for the binding to KcsA-KV1.1 or KcsA-KV1.3-bearing spheroplasts. (E,F) Competition between RFP-AgTx2 and different ligands for the binding to KcsA-KV1.1 or KcsA-KV1.3-bearing spheroplasts. Data of representative experiments are shown. Mean ± S.E. values are presented (number of cells per point, n ≥ 150).
	Figure 6. Application of FP-Tx in spheroplast binding assay as a screening tool.(A) Reversibility of eGFP-OSK1 and RFP-AgTx2 binding to KcsA-KV1.3 on spheroplast membrane. The “binding” bar shows signal from spheroplasts incubated with eGFP-OSK1 or RFP-AgTx2 (5.6 nM FP-Tx, 2 h, 37 °C), and the “dissociation” bar shows signal from the same spheroplasts incubated in a ligand-free medium for 4 h for FP-Tx wash-out. (B,C) Influence of potassium channel blockers charybdotoxin (ChTx, 300 nM), kaliotoxin (KTX, 30 nM), scyllatoxin (ScTx, 1 μM), and 4-aminopyridine (4-AP, 10 mM) and crude venoms (38 μg/ml) of M. eupeus, O. scrobiculosus, P. fasciata, and P. murinus on the binding of 5.6 nM eGFP-OSK1 (B) and RFP-AgTx2 (C) to KcsA-KV1.3-bearing spheroplasts. Mean ± S.E. values are shown (n = 3).

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