Journal article
Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape
The Journal of general physiology, Vol.150(7), pp.1017-1024
07/02/2018
DOI: 10.1085/jgp.201812075
PMCID: PMC6028492
PMID: 29866793
Abstract
Voltage-dependent activation of voltage-gated cation channels results from the outward movement of arginine-bearing helices within proteinaceous voltage sensors. The voltage-sensing residues in potassium channels have been extensively characterized, but current functional approaches do not allow a distinction between the electrostatic and steric contributions of the arginine side chain. Here we use chemical misacylation and in vivo nonsense suppression to encode citrulline, a neutral and nearly isosteric analogue of arginine, into the voltage sensor of the Shaker potassium channel. We functionally characterize the engineered channels and compare them with those bearing conventional mutations at the same positions. We observe effects on both voltage sensitivity and gating kinetics, enabling dissection of the roles of residue structure versus positive charge in channel function. In some positions, substitution with citrulline causes mild effects on channel activation compared with natural mutations. In contrast, substitution of the fourth S4 arginine with citrulline causes substantial changes in the conductance–voltage relationship and the kinetics of the channel, which suggests that a positive charge is required at this position for efficient voltage sensor deactivation and channel closure. The encoding of citrulline is expected to enable enhanced precision for the study of arginine residues located in crowded transmembrane environments in other membrane proteins. In addition, the method may facilitate the study of citrullination in vivo.
Details
- Title: Subtitle
- Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape
- Creators
- Daniel T Infield - Department of Molecular Physiology and Biophysics, The University of Iowa, Iowa City, IA, Iowa Neuroscience Institute, The University of Iowa, Iowa City, IAElizabeth E.L Lee - Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, ILJason D Galpin - Department of Molecular Physiology and Biophysics, The University of Iowa, Iowa City, IA, Iowa Neuroscience Institute, The University of Iowa, Iowa City, IAGrace D Galles - Department of Molecular Physiology and Biophysics, The University of Iowa, Iowa City, IA, Iowa Neuroscience Institute, The University of Iowa, Iowa City, IAFrancisco Bezanilla - Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, Institute for Biophysical Dynamics, The University of Chicago, Chicago, ILChristopher A Ahern - Department of Molecular Physiology and Biophysics, The University of Iowa, Iowa City, IA, Iowa Neuroscience Institute, The University of Iowa, Iowa City, IA
- Resource Type
- Journal article
- Publication Details
- The Journal of general physiology, Vol.150(7), pp.1017-1024
- DOI
- 10.1085/jgp.201812075
- PMID
- 29866793
- PMCID
- PMC6028492
- NLM abbreviation
- J Gen Physiol
- ISSN
- 0022-1295
- eISSN
- 1540-7748
- Grant note
- DOI: 10.13039/100000002, name: National Institutes of Health, award: R01GM106569, R24NS104617, R01GM030376; DOI: 10.13039/100000897, name: Cystic Fibrosis Foundation, award: INFIELD17F0
- Language
- English
- Date published
- 07/02/2018
- Academic Unit
- Molecular Physiology and Biophysics; Iowa Neuroscience Institute
- Record Identifier
- 9984070882702771
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