Why does the inner-helix mutation A413C double the stoichiometry of Kv1.3 channel block by emopamil but not by verapamil?

hKv1.3 channels in lymphocytes are targets for the chemotherapy treatment of autoimmune diseases. Phenylalkylamines block Kv1.3 channels by poorly understood mechanisms. In the inactivation-reduced mutant H399T, the second mutation A413C in S6 substantially decreases the potency of phenylalkylamines with a para-methoxy group at the phenylethylamine end, whereas potency of phenylalkylamines lacking this group is less affected. Intriguingly, completely demethoxylated emopamil blocks mutant H399T/A413C with a 2:1 stoichiometry. Here, we generated a triple mutant, H399T/C412A/A413C, and found that its emopamil-binding properties are similar to those of the double mutant. These data rule out disulfide bonding Cys412-Cys413, which would substantially deform the inner helix, suggest a clash of Cys413 with the para-methoxy group, and provide a distance constraint to dock phenylalkylamines in a Kv1.2-based homology model. Monte Carlo minimizations predict that the verapamil ammonium group donates an H-bond to the backbone carbonyl of Thr391 at the P-loop turn, the pentanenitrilephenyl moiety occludes the pore, whereas the phenylethylamine meta- and para-methoxy substituents approach, respectively, the side chains of Met390 and Ala413. In the double-mutant model, the Cys413 side chains accept H-bonds from two emopamil molecules whose phenyl rings fit in the hydrophobic intersubunit interfaces, whereas the pentanenitrilephenyl moieties occlude the pore. Because these interfaces are unattractive for a methoxylated phenyl ring, the ammonium group of respective phenylalkylamines cannot approach the Cys413 side chain and binds at the focus of P-helices, whereas the para-methoxy group clashes with Cys413. Our study proposes an atomistic mechanism of Kv1.3 block by phenylalkylamines and highlights the intra- and intersubunit interfaces as ligand binding loci.