title: Molecular determinants of KCNQ1 channel block by a benzodiazepine.
abstract: KCNQ1 channels underlie the slow delayed rectifier K+ current, mediate repolarization of cardiac action potentials, and are a potential therapeutic target for treatment of arrhythmia. (E)-(+)-N-[(3R)-2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl]-3-(2,4-dichlorophenyl)-2-propenamide [L-735821 (L-7)] is a potent blocker of KCNQ1 channels. Here we describe the structural determinants of KCNQ1 that are critical for high-affinity block by L-7 using site-directed mutagenesis to alter specific residues and voltage clamp to record channel currents in Xenopus laevis oocytes. Chimeric channels were constructed by combination of regions from L-7-sensitive KCNQ1 and L-7-insensitive KCNQ2 channel subunits. This approach localized the drug interaction site to the pore and S6 domains of KCNQ1. Substitution of single amino acids identified Thr-312 of the pore domain and Ile-337, Phe-339, Phe-340, and Ala-344 of the S6 domain as the most important molecular determinants of channel block. Some mutations also altered the inactivation properties of KCNQ1, but there was no correlation between extent of inactivation and sensitivity to block by L-7. Modeling was used to simulate the docking of L-7 to the KCNQ1 channel pore. The docking was consistent with our experimental data and predicts that L-7 blocks K+ conductance by physically precluding the occupancy of a K+ ion to a pore helix-coordinated site within the central hydrated cavity, a crucial step in ion permeation.
authors.affiliation: Department of Physiology, University of Utah, Salt Lake City, USA.
authors.forename: Guiscard
authors.initials: G
authors.lastname: Seebohm
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
authors.forename: Nathalie
authors.initials: N
authors.lastname: Strutz
authors.forename: Chris
authors.initials: C
authors.lastname: Culberson
authors.forename: Christian
authors.initials: C
authors.lastname: Lerche
authors.forename: Michael C
authors.initials: MC
authors.lastname: Sanguinetti
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001569:Benzodiazepines / Q000494:pharmacology*; D001665:Binding Sites; D004347:Drug Interactions; D006801:Humans; D051656:KCNQ Potassium Channels; D051657:KCNQ1 Potassium Channel; D051658:KCNQ2 Potassium Channel; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D015221:Potassium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
chemical_list: D051656:KCNQ Potassium Channels; D051657:KCNQ1 Potassium Channel; C496909:KCNQ1 protein, human; D051658:KCNQ2 Potassium Channel; C496914:KCNQ2 protein, human; C115921:L 735821; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011993:Recombinant Fusion Proteins; D001569:Benzodiazepines