K+ channel types targeted by synthetic OSK1, a toxin from Orthochirus scrobiculosus scorpion venom

Abstract

OSK1 (alpha-KTx3.7) is a 38-residue toxin cross-linked by three disulphide bridges that was initially isolated from the venom of the Asian scorpion Orthochirus scrobiculosus. OSK1 and several structural analogues were produced by solid-phase chemical synthesis, and were tested for lethality in mice and for their efficacy in blocking a series of 14 voltage-gated and Ca2+-activated K+ channels in vitro. In the present paper, we report that OSK1 is lethal in mice by intracerebroventricular injection, with a LD50 (50% lethal dose) value of 2 microg/kg. OSK1 blocks K(v)1.1, K(v)1.2, K(v)1.3 channels potently and K(Ca)3.1 channel moderately, with IC50 values of 0.6, 5.4, 0.014 and 225 nM respectively. Structural analogues of OSK1, in which we mutated positions 16 (Glu16-->Lys) and/or 20 (Lys20-->Asp) to amino acid residues that are conserved in all other members of the alpha-KTx3 toxin family except OSK1, were also produced and tested. Among the OSK1 analogues, [K16,D20]-OSK1 (OSK1 with Glu16-->Lys and Lys20-->Asp mutations) shows an increased potency on K(v)1.3 channel, with an IC50 value of 0.003 nM, without loss of activity on K(Ca)3.1 channel. These data suggest that OSK1 or [K16,D20]-OSK1 could serve as leads for the design and production of new immunosuppressive drugs.

Figure 1. Amino acid sequence (one-letter code) of OSK1 and comparison with those of other toxins belonging to the α-KTx3 structural family

(a) The amino acid sequences of scorpion toxins were aligned according to the positions of their half-cystine residues, numbered 1–6. The positions of half-cystine residues are highlighted in grey boxes. The asterisk indicates a C-terminal carboxylamidated extremity. Amino acid sequence identities (%) with OSK1 are indicated on the right. The position of OSK1 is indicated by an arrow. Basic and acidic residues are shown in bold and underlined italics, respectively. (b) Amino acid sequences of OSK1 and its chemically produced analogues. Mutated residues are shown in bold and numbered. The 3D solution structure of OSK1 can be found in the Protein Data Bank (code 1SCO) [1].

Figure 2. Synthesis and characterization of OSK1 and its analogues

(a) Analytical C₁₈ reversed-phase HPLC elution profile of crude reduced OSK1. Note the presence of non-peptide chemical scavengers in the elution profile (elution times approx. 15 min) that later ‘disappeared’ during oxidative folding, presumably by sticking to the plastic support. (b) Elution profile of crude folded/oxidized OSK1 after 72 h oxidation time. (c) Elution profile of folded/oxidized OSK1 after purification. (d) Deduced and experimental relative molecular masses (M+H)⁺ of synthetic OSK1 and analogues.

Figure 3. Structural analysis of OSK1 and its analogues by ¹H-NMR

(a) Contour plot of a NOESY spectrum of synthetic OSK1. Fingerprint and amide proton regions are shown on top and bottom, respectively. (b) One-dimensional ¹H-NMR spectra of OSK1 and its analogues. Only the representative amide proton regions are shown. Note that the overall distribution of resonance frequencies suggests that the 3D structures of all six peptides are similar.

Figure 4. Block of voltage-gated K_v1.1, K_v1.2, K_v1.3 and Ca²⁺-activated K_Ca3.1 channels by OSK1

(a) Original current traces in the absence (control) or presence of synthetic OSK1 at various concentrations. Top left, block of K_v1.1 current by OSK1. Top right, block of K_v1.2 current. Bottom left, block of K_v1.3 current. For each K_v-channel type, currents were elicited by depolarizing voltage steps from the holding potential −80 mV to +40 mV for 200 ms. Bottom right, OSK1-induced block of K_Ca3.1 current. Currents were elicited by 1 μM [Ca²⁺]_i and voltage ramps from the holding potential −80 mV to +40 mV for 400 ms. (b) Average normalized current inhibition by various concentrations of OSK1 for each channel type. Results are means±S.D. (n=4–5).

Figure 5. Activity of OSK1 analogues on K_v channels

(a) Average normalized inhibition of K_v1.1, K_v1.2, K_v1.3 and K_Ca3.1 currents by various concentrations of [K₁₆,D₂₀]-OSK1. Each data point corresponds to the mean±S.D. (n=4–5). (b) Average normalized inhibition of K_v1.1 currents by various concentrations of [P₁₂,K₁₆,D₂₀]-OSK1 (n=5) and comparison with OSK1 (n=5). (c) Average normalized inhibition of K_v1.2 currents by various concentrations of [P₁₂,K₁₆,D₂₀]-OSK1 (n=3). (d) Average normalized inhibition of K_v1.3 currents by various concentrations of [P₁₂,K₁₆,D₂₀]-OSK1 (n=5). (e) Current traces elicited at +40 mV for control condition or after application of 1 μM OSK1 (n=4) or 1.5 μM [K₁₆,D₂₀,Y₃₆]-OSK1 (n=5).

Figure 6. Comparison of amino acid sequences of pore domains of K_v1.3 with K_v1.1, K_v1.2 and K_Ca3.1 channels

Each K⁺ channel amino acid sequence is numbered and subdivided into structural and/or functional regions (transmembrane segments S5 and S6, turret region, pore helix and selectivity filter). Amino acid sequence identity with K_v1.3 channel is shaded grey. Amino acid residues predicted to interact with OSK1 are denoted by asterisks.

Figure 7. Functional maps of OSK1 on to K_v1.1, K_v1.2 and K_v1.3, and of [K₁₆,D₂₀]-OSK1 on to K_v1.3 channels

Theoretical functional maps of OSK1 and [K₁₆,D₂₀]-OSK1 depicting amino acid residues that are key for peptide interaction with K_v1.1, K_v1.2 or K_v1.3 channels. Interacting residues from the K_v channel are highlighted in red. I to IV before channel residue numbering specifies one of the four α-subunits forming a functional K_v channel. Colour codes: yellow (hydrophobic residues), light green (polar residues) and blue (basic residues). These maps were generated by docking simulations [37]. Swiss-Prot accession codes used are P16388 (mouse K_v1.1 channel), P16389 (human K_v1.2 channel) and P16390 (mouse K_v1.3 channel).