title: An allosteric mechanism for drug block of the human cardiac potassium channel KCNQ1.
abstract: The intracellular aspect of the sixth transmembrane segment within the ion-permeating pore is a common binding site for many voltage-gated ion channel blockers. However, the exact site(s) at which drugs bind remain controversial. We used extensive site-directed mutagenesis coupled with molecular modeling to examine mechanisms in drug block of the human cardiac potassium channel KCNQ1. A total of 48 amino acid residues in the S6 segment, S4-S5 linker, and the proximal C-terminus of the KCNQ1 channel were mutated individually to alanine; alanines were mutated to cysteines. Residues modulating drug block were identified when mutant channels displayed <50% block on exposure to drug concentrations that inhibited wild-type current by ≥90%. Homology modeling of the KCNQ1 channel based on the Kv1.2 structure unexpectedly predicted that the key residue modulating drug block (F351) faces away from the permeating pore. In the open-state channel model, F351 lines a pocket that also includes residues L251 and V254 in S4-S5 linker. Docking calculations indicated that this pocket is large enough to accommodate quinidine. To test this hypothesis, L251A and V254A mutants were generated that display a reduced sensitivity to blockage with quinidine. Thus, our data support a model in which open state block of this channel occurs not via binding to a site directly in the pore but rather by a novel allosteric mechanism: drug access to a side pocket generated in the open-state channel configuration and lined by S6 and S4-S5 residues.
authors.affiliation: John Oates Institute for Experimental Therapeutics, Departments of Medicine, Pharmacology and Center for Structural Biology, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Tao
authors.initials: T
authors.lastname: Yang
authors.forename: Jarrod A
authors.initials: JA
authors.lastname: Smith
authors.forename: Brenda F
authors.initials: BF
authors.lastname: Leake
authors.forename: Charles R
authors.initials: CR
authors.lastname: Sanders
authors.forename: Jens
authors.initials: J
authors.lastname: Meiler
authors.forename: Dan M
authors.initials: DM
authors.lastname: Roden
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000494:Allosteric Regulation / Q000187:drug effects*; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D006321:Heart / Q000187:drug effects*; D006801:Humans; D051657:KCNQ1 Potassium Channel / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009206:Myocardium / Q000378:metabolism*; D011802:Quinidine / Q000378:metabolism
chemical_list: D051657:KCNQ1 Potassium Channel; C496909:KCNQ1 protein, human; D011802:Quinidine; D003545:Cysteine; D000409:Alanine