title: Unnatural amino acid photo-crosslinking of the IKs channel complex demonstrates a KCNE1:KCNQ1 stoichiometry of up to 4:4.
abstract: Cardiac repolarization is determined in part by the slow delayed rectifier current (IKs), through the tetrameric voltage-gated ion channel, KCNQ1, and its β-subunit, KCNE1. The stoichiometry between α and β-subunits has been controversial with studies reporting either a strict 2 KCNE1:4 KCNQ1 or a variable ratio up to 4:4. We used IKs fusion proteins linking KCNE1 to one (EQ), two (EQQ) or four (EQQQQ) KCNQ1 subunits, to reproduce compulsory 4:4, 2:4 or 1:4 stoichiometries. Whole cell and single-channel recordings showed EQQ and EQQQQ to have increasingly hyperpolarized activation, reduced conductance, and shorter first latency of opening compared to EQ - all abolished by the addition of KCNE1. As well, using a UV-crosslinking unnatural amino acid in KCNE1, we found EQQQQ and EQQ crosslinking rates to be progressively slowed compared to KCNQ1, which demonstrates that no intrinsic mechanism limits the association of up to four β-subunits within the IKs complex.
authors.affiliation: Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, Canada.
authors.forename: Christopher I
authors.initials: CI
authors.lastname: Murray
authors.affiliation: Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, Canada.
authors.forename: Maartje
authors.initials: M
authors.lastname: Westhoff
authors.affiliation: Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, Canada.
authors.forename: Jodene
authors.initials: J
authors.lastname: Eldstrom
authors.affiliation: Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, Canada.
authors.forename: Emely
authors.initials: E
authors.lastname: Thompson
authors.affiliation: Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, Canada.
authors.forename: Robert
authors.initials: R
authors.lastname: Emes
authors.affiliation: Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, Canada.
authors.forename: David
authors.initials: D
authors.lastname: Fedida
mesh_terms: D006801:Humans; D051657:KCNQ1 Potassium Channel / Q000737:chemistry* / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated / Q000737:chemistry* / Q000378:metabolism*; D021122:Protein Subunits / Q000032:analysis*
chemical_list: C489622:KCNE1 protein, human; D051657:KCNQ1 Potassium Channel; C496909:KCNQ1 protein, human; D024642:Potassium Channels, Voltage-Gated; D021122:Protein Subunits
keywords: KCNE1; KCNQ1; biochemistry; biophysics; human; photo-crosslinking; single channel; stoichiometry; structural biology; unnatrual amino acid