Atomic mutagenesis in ion channels with engineered stoichiometry

John D Lueck; Adam L Mackey; Daniel T Infield; Jason D Galpin; Jing Li; Benoît Roux; Christopher A Ahern

doi:10.7554/eLife.18976

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Atomic mutagenesis in ion channels with engineered stoichiometry

Journal article

Open access

Peer reviewed

Atomic mutagenesis in ion channels with engineered stoichiometry

John D Lueck, Adam L Mackey, Daniel T Infield, Jason D Galpin, Jing Li, Benoît Roux and Christopher A Ahern

eLife, Vol.5, e18976

10/06/2016

DOI: 10.7554/eLife.18976

PMCID: PMC5092047

PMID: 27710770

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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.

Gene Expression

Mutation

Protein Engineering

Thermodynamics

Humans

Shaker Superfamily of Potassium Channels - metabolism

Mutagenesis, Site-Directed - methods

NAV1.4 Voltage-Gated Sodium Channel - metabolism

Recombinant Fusion Proteins - metabolism

Oocytes - cytology

Shaker Superfamily of Potassium Channels - chemistry

HEK293 Cells

Protein Domains

Binding Sites

Amino Acid Sequence

Protein Conformation, alpha-Helical

NAV1.4 Voltage-Gated Sodium Channel - chemistry

NAV1.4 Voltage-Gated Sodium Channel - genetics

Shaker Superfamily of Potassium Channels - genetics

Xenopus laevis

Models, Molecular

Recombinant Fusion Proteins - chemistry

Membrane Potentials - physiology

Molecular Dynamics Simulation

Patch-Clamp Techniques

Animals

Oocytes - physiology

Protein Binding

Recombinant Fusion Proteins - genetics

Kinetics

Ion Channel Gating

Details

Title: Subtitle: Atomic mutagenesis in ion channels with engineered stoichiometry
Creators: John D Lueck - Department of Molecular Physiology and Biophysics, The University of Iowa, Iowa City, United States
Adam L Mackey - Department of Molecular Physiology and Biophysics, The University of Iowa, Iowa City, United States
Daniel T Infield - Department of Molecular Physiology and Biophysics, The University of Iowa, Iowa City, United States
Jason D Galpin - Department of Molecular Physiology and Biophysics, The University of Iowa, Iowa City, United States
Jing Li - University of Chicago
Benoît Roux - Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, United States
Christopher A Ahern - Department of Molecular Physiology and Biophysics, The University of Iowa, Iowa City, United States
Resource Type: Journal article
Publication Details: eLife, Vol.5, e18976
DOI: 10.7554/eLife.18976
PMID: 27710770
PMCID: PMC5092047
NLM abbreviation: Elife
ISSN: 2050-084X
eISSN: 2050-084X
Publisher: England
Grant note: U54 GM087519 / NIGMS NIH HHS R01 GM106569 / NIGMS NIH HHS R01 GM062342 / NIGMS NIH HHS
Language: English
Date published: 10/06/2016
Academic Unit: Molecular Physiology and Biophysics; Iowa Neuroscience Institute
Record Identifier: 9984070820802771

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