title: Atomic mutagenesis in ion channels with engineered stoichiometry.
abstract: C-type inactivation of potassium channels fine-tunes the electrical signaling in excitable cells through an internal timing mechanism that is mediated by a hydrogen bond network in the channels' selectively filter. Previously, we used nonsense suppression to highlight the role of the conserved Trp434-Asp447 indole hydrogen bond in Shaker potassium channels with a non-hydrogen bonding homologue of tryptophan, Ind (Pless et al., 2013). Here, molecular dynamics simulations indicate that the Trp434Ind hydrogen bonding partner, Asp447, unexpectedly 'flips out' towards the extracellular environment, allowing water to penetrate the space behind the selectivity filter while simultaneously reducing the local negative electrostatic charge. Additionally, a protein engineering approach is presented whereby split intein sequences are flanked by endoplasmic reticulum retention/retrieval motifs (ERret) are incorporated into the N- or C- termini of Shaker monomers or within sodium channels two-domain fragments. This system enabled stoichiometric control of Shaker monomers and the encoding of multiple amino acids within a channel tetramer.
authors.affiliation: Department of Molecular Physiology and Biophysics, The University of Iowa, Iowa City, United States.
authors.forename: John D
authors.initials: JD
authors.lastname: Lueck
authors.affiliation: Department of Molecular Physiology and Biophysics, The University of Iowa, Iowa City, United States.
authors.forename: Adam L
authors.initials: AL
authors.lastname: Mackey
authors.affiliation: Department of Molecular Physiology and Biophysics, The University of Iowa, Iowa City, United States.
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Infield
authors.affiliation: Department of Molecular Physiology and Biophysics, The University of Iowa, Iowa City, United States.
authors.forename: Jason D
authors.initials: JD
authors.lastname: Galpin
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, United States.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, United States.
authors.forename: Benoît
authors.identifier: 0000-0002-5254-2712
authors.initials: B
authors.lastname: Roux
authors.affiliation: Department of Molecular Physiology and Biophysics, The University of Iowa, Iowa City, United States.
authors.forename: Christopher A
authors.identifier: 0000-0002-7975-2744
authors.initials: CA
authors.lastname: Ahern
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D062553:NAV1.4 Voltage-Gated Sodium Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072417:Protein Domains; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051637:Shaker Superfamily of Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014982:Xenopus laevis
chemical_list: D062553:NAV1.4 Voltage-Gated Sodium Channel; D011993:Recombinant Fusion Proteins; C106251:SCN4A protein, human; D051637:Shaker Superfamily of Potassium Channels
keywords: C-type inactivation; atomic mutagenesis; biochemistry; biophysics; intein protein ligation; molecular dynamics; noncanonical amino acids; structural biology; voltage-gated sodium channels; xenopus