Potassium Channel Block by a Tripartite Complex of Neutral Ligands with a Potassium Ion

K + channels are blocked by structurally diverse compounds. While hydrophilic cations like TEA block Kv channels with a stoichiometry of 1:1, many uncharged lipophilic compounds like the novel immunosuppressant PAP‐1 (Kv1.3 IC 50 2 nM) exhibit Hill coefficients of 2. To determine the mechanism of PAP‐1 block, we first explored the SAR around PAP‐1 and found that the coumarin ring carbonyl group is indispensable, but does not accept an H‐bond from the channel. We next demonstrated that block by PAP‐1 is voltage‐dependent, a feature expected for cationic but not neutral ligands. Through molecular modeling we then proposed a model in which the carbonyl groups of two PAP‐1 molecules coordinate a K + ion in the permeation pathway, while the hydrophobic phenoxyalkoxy side‐chains extend into the intrasubunit interfaces between helices S5 and S6 and reach the L45 linker. To test the model we generated 58 point mutants and then determined their biophysical properties and their sensitivity to PAP‐1. We found excellent agreement between the atomic‐scale model and the experimental studies. Besides the known drug‐binding locus in the inner pore, which is rather conserved between different Kv channels, the PAP‐1 receptor involves low homology loci. These loci constitute attractive targets for the design of subtype‐specific K + channel drugs and offer new directions for structure‐based drug design. Supported by CIHR, NIH, and HHMI.