Designer peptide CmTx potently blocks Kv1.5 channels by neutralizing R487 arginines, revealing electrostatic complementation

Voltage-gated potassium (Kv) channels are crucial drug targets, but Kv1.5 specifically resists inhibition by common peptide neurotoxins. This resistance stems from four positively-charged arginines (R487) on each pore-forming subunit, which electrostatically repel typical pore-blocking toxins. The slow-inactivated conformation of Kv1.5, promoted by rapid firing and acidosis, is implicated in conditions like atrial fibrillation and ischemia, making its inhibition a challenging yet important therapeutic goal.

Researchers isolated chimera toxin (CmTx), a de novo peptide engineered on a SAK1 scaffold, using cell-based phage-display library panning. They assessed its potency against Kv1.5, determining a Ki of 127 nM, and evaluated its selectivity against seven other Kv subtypes. Scanning mutagenesis was employed to identify critical binding residues on both CmTx and Kv1.5. Finally, AlphaFold modeling was utilized to elucidate the molecular mechanism by which CmTx interacts with and inhibits the Kv1.5 channel.

CmTx potently and selectively blocked the Kv1.5 channel with a Ki of 127 nM, demonstrating a preference for the slow-inactivated conformation associated with atrial fibrillation and ischemia. CmTx showed minimal inhibition of seven other Kv subtypes, highlighting its impressive selectivity. Scanning mutagenesis revealed specific CmTx and Kv1.5 residues crucial for binding. AlphaFold modeling elucidated that acidic residues on CmTx electrostatically neutralize the four positively-charged R487 arginines on the Kv1.5 pore-forming subunits. This complementary charge interaction allows CmTx to overcome the channel's inherent electrostatic repulsion mechanism and effectively occlude the pore, establishing a novel electrostatic pore-blocking mechanism.