title: Why does the inner-helix mutation A413C double the stoichiometry of Kv1.3 channel block by emopamil but not by verapamil?
abstract: 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.
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada.
authors.forename: Alexey
authors.initials: A
authors.lastname: Rossokhin
authors.forename: Tobias
authors.initials: T
authors.lastname: Dreker
authors.forename: Stephan
authors.initials: S
authors.lastname: Grissmer
authors.forename: Boris S
authors.initials: BS
authors.lastname: Zhorov
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D051664:Kv1.3 Potassium Channel / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D026902:Potassium Channel Blockers / Q000378:metabolism / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D013237:Stereoisomerism; D014700:Verapamil / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D051664:Kv1.3 Potassium Channel; D026902:Potassium Channel Blockers; D014700:Verapamil; D003545:Cysteine; C052752:emopamil; D000409:Alanine