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

Abstract

Potassium channels are targets for medically important drugs of large chemical diversity. While classical hydrophilic cations like tetraethylammonium block Kv channels with a stoichiometry of 1:1, many uncharged lipophilic (neutral) compounds exhibit Hill coefficients of 2. An example is the alkoxypsoralen PAP-1, which blocks Kv1.3 channels in lymphocytes with an IC50 of 2 nM and constitutes a potential drug for autoimmune disease such as type-1 diabetes, multiple sclerosis, rheumatoid arthritis, and psoriasis. The atomic mechanism of Kv channel block by ligands like PAP-1 is unknown. We first studied structure-activity relationships of PAP-1 derivatives and found that the carbonyl group in PAP-1's coumarin ring 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. We then employed molecular modeling to predict the PAP-1 receptor and arrived at a model in which the carbonyl groups of two PAP-1 molecules coordinate a potassium 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. We tested the model by generating 58 point mutants involving residues in and around the predicted receptor, and then determined their biophysical properties and sensitivity to block by PAP-1. We found excellent agreement between the atomistic model and the results of 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 loci where sequence homology is low. These loci constitute attractive targets for the design of subtype-specific potassium channel drugs and offer new directions for structure-based drug design.Supported by NSERC, CIHR, NIH, and HHMI.