Voltage-gated sodium (NaV)- and potassium (KV)-channel proteins underlie the regulation of basal electrical excitability and the initiation and repolarization of action potentials in virtually all excitable cells. These channels have evolved from primordial K+-selective pores into diverse proteins with regulatory mechanisms that enable them to respond to specific stimuli in the nervous, cardiovascular, and immune systems. The importance of this protein family to human health is highlighted by the facts that: inherited or acquired defects in NaV or Kv channels cause epilepsy, myotonia, erythromelalgia and cardiac arrhythmias;mutations that lead to changes in the gating kinetics or expression of K+ channels in excitable tissues such as cardiac muscle can lead to arrhythmias and susceptibility to sudden cardiac death (long or short QT syndromes);and significant electrical remodeling of KV- and NaV-channel expression is observed during cardiac hypertrophy or persistent arrhythmias. Unfortunately, a broad spectrum of excitability disorders remains largely untreatable, and a fresh approach to closing the gap in our understanding of NaV and KV gating and selectivity will be needed if effective therapeutics are to be developed. Notably, although ion channels (particularly those of the voltage-gated ion channel superfamily) have been characterized on both the macroscopic and atomic levels, these studies lack the resolution needed to identify essential chemical property(s) of the amino acids that have been implicated in functional roles. The lack of such information remains a significant block to our understanding of ion permeation and channel gating, and ultimately, effective drug design. Here we propose to design and apply powerful synthetic tools, in the form of tailor-made unnatural amino acids, as a means of achieving hypothesis-driven atomic-level mutagenesis to reach a physiological endpoint: an understanding of the basis of ion selectivity and channel gating. Further, although we expect to eventually be able to perform structure-based drug design, no structures yet exist for eukaryotic NaVs, and the work proposed here will inform us directly about which traits of bacterial NaV's (where structures are now available) are relevant to eukaryotic NaV's. Finally, the results of our study will remove a significant technical barrier to an atomic-level, functional understanding of the gating and permeation mechanisms employed by NaV and KV channels-two proven drug targets in the management of excitability disorders. Success of the proposed study will make it possible to generate novel amino acids that will be widely available to the research community.

Public Health Relevance

The results of our study will remove a significant technical barrier that has barred an atomic-level, functional understanding of sodium and potassium channels-two proven drug targets in the management of excitability disorders such as epilepsy, cardiac arrhythmia and pain. Success of the proposed study will enable the generation of powerful research tools that will be widely available to the research community.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM106569-02
Application #
8731952
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Fabian, Miles
Project Start
2013-09-15
Project End
2018-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
2
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Iowa
Department
Physiology
Type
Schools of Medicine
DUNS #
City
Iowa City
State
IA
Country
United States
Zip Code
52242
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Thompson, Ammon; Infield, Daniel T; Smith, Adam R et al. (2018) Rapid evolution of a voltage-gated sodium channel gene in a lineage of electric fish leads to a persistent sodium current. PLoS Biol 16:e2004892
Infield, Daniel T; Lee, Elizabeth E L; Galpin, Jason D et al. (2018) Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape. J Gen Physiol 150:1017-1024
Infield, Daniel T; Lueck, John D; Galpin, Jason D et al. (2018) Orthogonality of Pyrrolysine tRNA in the Xenopus oocyte. Sci Rep 8:5166
Steinberg, Ximena; Kasimova, Marina A; Cabezas-Bratesco, Deny et al. (2017) Conformational dynamics in TRPV1 channels reported by an encoded coumarin amino acid. Elife 6:
Leisle, Lilia; Chadda, Rahul; Lueck, John D et al. (2016) Cellular encoding of Cy dyes for single-molecule imaging. Elife 5:
Tian, Yutao; Aursnes, Marius; Hansen, Trond Vidar et al. (2016) Atomic determinants of BK channel activation by polyunsaturated fatty acids. Proc Natl Acad Sci U S A 113:13905-13910
Ahern, Christopher A; Payandeh, Jian; Bosmans, Frank et al. (2016) The hitchhiker's guide to the voltage-gated sodium channel galaxy. J Gen Physiol 147:1-24
Lueck, John D; Mackey, Adam L; Infield, Daniel T et al. (2016) Atomic mutagenesis in ion channels with engineered stoichiometry. Elife 5:
Pless, Stephan A; Kim, Robin Y; Ahern, Christopher A et al. (2015) Atom-by-atom engineering of voltage-gated ion channels: magnified insights into function and pharmacology. J Physiol 593:2627-34

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