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

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.