Localization and Molecular Determinants of the Hanatoxin Receptors on the Voltage-Sensing Domains of a K⁺ Channel

Hanatoxin inhibits voltage-gated K⁺ channels by modifying the energetics of activation. We studied the molecular determinants and physical location of the Hanatoxin receptors on the drk1 voltage-gated K⁺ channel. First, we made multiple substitutions at three previously identified positions in the COOH terminus of S3 to examine whether these residues interact intimately with the toxin. We also examined a region encompassing S1–S3 using alanine-scanning mutagenesis to identify additional determinants of the toxin receptors. Finally, guided by the structure of the KcsA K⁺ channel, we explored whether the toxin interacts with the peripheral extracellular surface of the pore domain in the drk1 K⁺ channel. Our results argue for an intimate interaction between the toxin and the COOH terminus of S3 and suggest that the Hanatoxin receptors are confined within the voltage-sensing domains of the channel, at least 20–25 Å away from the central pore axis.