Novel Scorpion Toxin ω-Buthitoxin-Hf1a Selectively Inhibits Calcium Influx via CaV3.3 and CaV3.2 and Alleviates Allodynia in a Mouse Model of Acute Postsurgical Pain

Abstract

Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type Ca_V3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type Ca_Vs but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH₂) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH₂ to be a concentration-dependent partial inhibitor of Ca_V3.2 (IC₅₀ = 1.18 μM) and Ca_V3.3 (IC₅₀ = 0.49 μM) depolarized currents but was ineffective at Ca_V3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type Ca_V2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH₂ was determined using NMR spectroscopy and used in docking studies to predict its binding site at Ca_V3.2 and Ca_V3.3. As both Ca_V3.2 and Ca_V3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH₂ was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH₂ produced antiallodynia in both mechanical and thermal pain.

Keywords: T-type calcium channels; acute post-surgical pain; peripheral pain; venom peptides.

Figure 1

(a) RP-HPLC trace of H. franzwerneri fractions (indicated in green points) I and II and their maximum responses at Ca_V3.2 in the FLIPR assay; (b) Response over baseline traces of the two fractions I and II isolated from scorpion H. franzwerneri, showing 72.4% and 66.2% inhibition of Ca²⁺ responses in Ca_V3.2, respectively.

Figure 2

Co-elution of the native and synthetic peptides. (a) Co-elution of native fraction I with synthetic Hf1a-NH₂. (b) Co-elution of native fraction II with synthetic Hf1a-OH.

Figure 3

The NMR structure of the C-terminally amidated ω-Buthitoxin-Hf1a. (a) Superposition of the 20 lowest energy structures of ω-Buthitoxin-Hf1a, with cysteine shown in yellow, histidine in blue, asparagine in cyan, threonine in orange, proline in light green, serine in pink, and tryptophane in green. (b) The lowest energy NMR solution structure of the ω-Buthitoxin-Hf1a backbone.

Figure 4

Effect of Hf1a-NH₂ on Ca_V3.1, Ca_V3.2, and Ca_V3.3 current. (a) Hf1a-NH₂ significantly inhibited (6.6%) the recombinant hCa_V3.1 channel current at 25 μM (p < 0.05; n = 4), while lower concentrations were ineffective; (b) representative I_Ca during 200 ms depolarizations to V_max (−20 mV) from a holding potential of −90 mV before and after perfusions of 25 µM of Hf1a-NH₂, as indicated. (c) Concentration response curve of Hf1a-NH₂ on recombinant hCa_V3.2 channels with 31.7% current inhibition at 25 μM (n = 4); (d) representative I_Ca during 200 ms depolarizations to V_max (−25 mV) from a holding potential of −90 mV before and after perfusions of 0.024 µM and 25 µM of Hf1a-NH₂, as indicated. (e) Concentration response curve of Hf1a-NH₂ on recombinant hCa_V3.3 channels with 44.6% current inhibition at 25 μM (n = 4); (f) representative I_Ca during 200 ms depolarizations to V_max (−10 mV) from a holding potential of −90 mV before and after perfusions of 0.024 µM and 1.56 µM of Hf1a-NH₂, as indicated. Data are means ± SEM.

Figure 5

Representative N-type (a) and L-type (b) Ca²⁺ response over baseline traces with addition of Hf1a_NH₂ (0.781–200 µM); and N-type (c) and L-type (d) Ca²⁺ response over baseline traces with addition of Hf1a_OH (0.781–200 µM).

Figure 6

Predicted binding mode of Hf1a (colored red) in human Ca_V3.2 and Ca_V3.3 (colored green). (a) General view of the lowest energy docking pose of Hf1a binding to the Ca_V3.2 central cavity of the Ca²⁺ permeation pore under the selectivity filter of the channel; local view highlights the interactions of His1 from Hf1a with Glu378 and Gln973 in Ca_V3.2, whereas Thr3, His8, and Trp10 showed polar interaction with Gln973, Lys1503, and Gln1848, respectively. (b) General view of the lowest energy docking pose of Hf1a binding to the Ca_V3.3 central cavity of the Ca²⁺ permeation pore under the selectivity filter of the channel, which is the published binding site of pimozide (colored cyan) in Ca_V3.3; local view highlights the interactions of His1 of Hf1a with Lys1379 and Thr1676 in Ca_V3.3, and interactions of Asn7 with Ser1419 and Lys1379. Other significant interactions were found between Thr3-Gln822, Asn5-Ile390, and Trp10-Gln1718. Predicted hydrogen bonds are shown as yellow dashed lines with all distances less than 4 Å.

Figure 7

Behavioral characterization of a mouse model of incision-induced acute post-surgical pain. Photographs of crucial steps of incisional surgery (a–c) and sham surgery (d,e) on the mouse hind paw. (a) A 7-mm longitudinal incision is made through the glabrous skin and fascia of the plantar surface of the right hind paw using a number 11 sterile surgical scalpel. The incision started about 3 mm from the proximal edge of the heel and extended toward the toes. (b) The underlying flexor digitorum brevis muscle is elevated to mimic muscle retraction and incised longitudinally with its origin and insertion intact. (c) The wound was closed with two sterile sutures after hemostasis, using the simple interrupted suture technique. (d) Two sterile sutures were carefully stitched into the skin during anesthesia in sham surgery. (e) The suturing area was disinfected with 5% povidone–iodine solution. Behavioral tests (mechanical and thermal) to characterize the mouse model of post-surgical pain (f,g). (f) Mechanical allodynia is present 24 h post-surgery with a significantly reduced paw withdrawal threshold in the surgery group compared to the sham group and compared to pre-surgery controls (n = 12 per group; **** p < 0.0001; one-way ANOVA with Dunnett’s multiple comparisons test). (g) Thermal allodynia is present 24 h post-surgery with a significantly reduced paw withdrawal latency time in the surgery group compared to the sham group and compared to pre-surgery controls (n = 12 per group; **** p < 0.0001; one-way ANOVA with Dunnett’s multiple comparisons test).

Figure 8

Analgesic effects of Hf1a-NH₂ on incision-induced mechanical and thermal allodynia, assessed 24 h post-surgery in mice. (a) Compared with vehicle control, the paw withdrawal thresholds of post-surgery mice increased significantly after intraplantar injection of 0.1 and 0.2 nmol/paw (5 μM and 10 μM; 20 μL) Hf1a-NH₂ and 6.0 pmol/paw (300 nM; 20 μL) control peptide MVIIA (* p < 0.05; **** p < 0.0001; **** p < 0.0001, respectively; n = 6 per group); (b) Compared with vehicle control, the paw withdrawal latency time of post-surgery mice increased significantly after intraplantar injection of 0.02, 0.1, and 0.2 nmol/paw (1 μM, 5 μM and 10 μM; 20 μL) Hf1a-NH₂ and 6.0 pmol/paw (300 nM; 20 μL) control peptide MVIIA (* p < 0.05; **** p < 0.0001; **** p < 0.0001; ** p < 0.01, respectively; n = 6 per group); Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test.