Voltage-sensor conformational microenvironments encode isoform-selective ion channel activation.

Isoform-selective opening of voltage-gated potassium (Kv) channels is desirable for its therapeutic potential yet mechanistically little-understood. Rosemary leaf metabolite carnosic acid (CA) is a high-efficacy opener of the neuronal Kv7.3 channel but exerts minimal effects on Kv7.2 or Kv7.2/3 heteromers. Here, using alanine-scanning mutagenesis, electrophysiology, radioligand binding, and all-atom molecular dynamics simulations, we found that CA achieves high-efficacy activation of Kv7.3 by binding at the extracellular cap of the voltage-sensing domain (VSD), independent of the canonical retigabine pore binding site. Kv7.3 samples a binding-competent VSD conformation absent in Kv7.2, characterized by an expanded extracellular pocket and a favorable electrostatic environment. Central to this mechanism, Kv7.3-L226 functions as a hydrophobic latch that tunes the energetic landscape of the voltage sensor to control ligand efficacy. Unexpectedly, CA potentiates Kv7.2/3 activation by retigabine via ligand-initiated positive allosteric coupling between the VSD and pore, thus enhancing the anticonvulsant action of retigabine despite CA lacking standalone anticonvulsant activity. The findings establish a mechanistic framework in which isoform selectivity arises from VSD conformational microenvironments rather than canonical binding determinants and highlight the voltage sensor as a tunable target for developing selective Kv channel openers.