Cross Pharmacological, Biochemical and Computational Studies of a Human Kv3.1b Inhibitor from Androctonus australis Venom

Figures

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  Figure 1. Inhibitory effect of AahG50 on Kv3.1 currents. (Aa)) Purification of Androctonus australis hector scorpion venom. Dialyzed venom loaded into a Sephadex G50 column allowed the collect 5 fractions (M1, M2, AahG50, M3 and M4). (Ab)) Dose–response curve of Kv3.1 channel inhibition at various concentrations of AahG50. Current amplitudes measured at the end of the depolarizing pulse (+60 mV), the percentage of inhibition plotted against respective concentrations of AahG50 (10, 25, 50, 100 and 150 µg/mL). (Ba)) Whole-cell currents, recorded in Xenopus oocytes, elicited by the application of 250 ms depolarizing pulses from −120 mV to +70 mV in 10 mV increments, from a holding potential of −80 mV, under control conditions and after the addition of 50 µg/mL AahG50. (Bb)) The normalized current voltage relationship of Kv3.1 currents in Xenopus oocytes plotted under control conditions and after the perfusion of 50 µg/mL of AahG50. (Ca)) Whole-cell currents, recorded in CHO cells, elicited by the application of 250 ms depolarizing pulses from −120 mV to +60 mV in 20 mV increments, from a holding potential of −80 mV, under control conditions and after the addition of 50 µg/mL AahG50. (Cb)) The…

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  Figure 2. Identification of FPLC subfractions in AahG50 active on Kv3.1 channel. (Aa) Purification of AahG50 with FPLC. Seven milligrams of proteins were loaded per run on a cation exchange Resource S column pre-equilibrated with 0.05 M ammonium acetate buffer (pH 6.6). Proteins were eluted with a 40 min linear gradient from 0.05 to 0.5 M ammonium acetate (pH 6.6) at a flow rate of 1 mL/min; absorbance was monitored at 280 nm. (Ab) Dose–response curve for the inhibition of Kv3.1 by various concentrations of F5. Current amplitudes were measured at the end of the depolarizing pulse (+70 mV), the percentage of inhibition was plotted against respective concentrations of F5. The curve was fitted by the Hill equation. (Ba) Whole-cell currents, recorded in Xenopus oocytes, elicited by the application of 250 ms depolarizing pulses from −120 mV, to +70 mV in 10 mV increments, from a holding potential of −80 mV, under control conditions and after the addition of 5 µg/mL of F5 fraction. (Bb) The normalized current voltage relationship of Kv3.1 currents in Xenopus oocytes were plotted under control conditions and after the perfusion of 5 µg/mL of F5. (Ca) Whole-cell currents, recorded in CHO cells,…

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  Figure 3. Characterization of H7 inhibitory effect on Kv3.1. (Aa) HPLC purification of AahG50 venom fraction (F5) obtained with FPLC method. Fifty micrograms of F5 were loaded per run on a C18 column reversed phase HPLC and 18 fractions (H1 to H18) eluted at a flow rate of 1 mL/min, using a multi-step gradient (90 min) from 0 to 60% of buffer B (0.1% TFA in CH3CN) in buffer A (0.1% TFA in water). (Ab) Kv3.1 normalized current at +60 mV shown before, and after perfusion of H7 at 0.5 and 1 µg/mL and after washing. (Ba) Whole-cell currents, recorded in mammalian cells, elicited by the application of 250 ms depolarizing pulses from −120 mV, to +60 mV in 20 mV increments, from a holding potential of −80 mV, under control conditions and after the addition of 1µg/mL of H7. (Bb) The normalized conductance (G) voltage relationship of Kv3.1 current plotted under control conditions and after the perfusion of 1 µg/mL of H7.

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  Figure 4. Validation of H7 inhibitory effect on Kv3.1 single current. (A) A representative trace of Kv3.1 channels in planar lipid bilayer from 4 min of continuous recording at −60 mV. Closed (c) and open (o) levels are indicated. H7 (0.6 µg/mL) is added at the indicated time point. (B) Fits of Gaussian distributions of multiple histograms led to a single channel current level of 4.74 ± 0.063 pA at −60 mV. (C) Dwell time of the whole 4 min recording at −60 mV revealed channel conductance levels (levels 1 to 8). (D,E) represent, respectively multiple histograms and dwell time after addition of H7.

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  Figure 5. NanoLC/HRMS-MS analysis of H7. (A) Purification of H7 subfraction. The purification of fraction H7 was repeated on a C18 column with a modified gradient allowing to collect a unique sharp peak eluted at 38 min. (B) H7 fraction was subjected to disulfide bridges reduction and trypsin digestion, and the obtained peptide mixture was analysed by NanoLC/HRMS-MS. Data analysis led to the identification in the H7 fraction of Aah6, Beta-insect excitatory 1 OS, Aah2, Aah3, Neurotoxin-like protein STR 1, G-TIOS, alpha-KTx 15.1 and AaTXK-beta. Signal peptide and polypeptide sequences are not reported.

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  Figure 6. Molecular modeling of alpha-KTx 15.1 and Kv3.1 channel interactions. (A) 3-D structure model of alpha-KTx 15.1 adopts the common Csαβ fold that allows the molecule to have a stable three-dimensional conformation. alpha-KTx 15.1 sequence (37 amino acids), alpha-helix coloured in red, beta sheet in yellow, cysteine residues in green. (B) Hot spot amino acids (coloured) implicated in the interaction between alpha-KTx 15.1 (a) and Kv3.1 channel (b). (C) Docking complex of alpha-KTx 15.1/Kv3.1 showing that the beta sheet of the toxin interacts with the selectivity filter residues of the channel. The pore is physically blocked with the K27 lateral chain of alpha-KTx 15.1.