A novel spider toxin as a selective antagonist of the Kv1 subfamily of voltage-gated potassium channels through gating modulation.

Members of the voltage-gated potassium channel subfamily (Kv1) are essential for the nervous and immune systems, necessitating novel modulators and deeper insights into their structure-function relationships. While all known peptide inhibitors targeting Kv1 channels are pore blockers, we identified MrVIII (κ-HxTx-MrVIII), a novel peptide toxin from the venom of spider Macrothele raveni, as the first voltage-gating modifier antagonist with selective activity against Kv1 channels. MrVIII exhibits high-affinity inhibition of Kv1.2, Kv1.3, Kv1.5, and Kv1.6, completely suppressing their currents. By contrast, it selectively inhibits the initial activation phase of Kv1.1, Kv1.4, and Kv1.7 with lower affinity, reflecting its differential subtype modulation. Gating current analyses revealed that MrVIII stabilizes the voltage sensor of Kv1 channels in its resting state, thereby preventing activation upon depolarization. The interaction between MrVIII and Kv1.1, Kv1.4, and Kv1.7 is unstable, with the voltage sensor of Kv1.7, initially stabilized in the resting state by the toxin, potentially transitioning back to an activated state, influenced by the strength and duration of depolarization. Alanine-scanning mutagenesis identified the S3-S4 region as the critical interaction region, with the conserved residue Y339 (in Kv1.3) serving as a key binding site across subtypes. Additionally, the contribution of E283 and T286 in Kv1.1 and A256 in Kv1.7 are key residues in defining channel's pattern in inhibition by MrVIII, compared to Kv1.3. These findings establish MrVIII as a valuable molecular tool for studying Kv1 channels, offering potential pathways for drug development and therapeutic applications.