Asymmetric functional contributions of acidic and aromatic side chains in sodium channel voltage-sensor domains

Pless, Stephan A.; Elstone, Fisal D.; Nićiforović, Ana; Galpin, Jason D.; Yang, Runying; Kurata, Harley T.; Ahern, Christopher A.

doi:10.1085/jgp.201311036

Cited by 42 publications

(44 citation statements)

References 60 publications

(116 reference statements)

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“…Surprisingly, the standard ligation conditions (37°C, 40 min) used for nonsense suppression by our lab and others (Pantoja et al, 2009; Pless et al, 2013, 2014; Nowak et al, 1998) resulted in relatively poor tRNA acylation yields with Cy-ncAAs (Figure 2A). We reasoned that this low ligation efficiency could, in part, be due to hydrolysis of the Cy-ncAA from the tRNA occurring during the enzymatic ligation reaction at 37°C; hydrolysis is a highly temperature sensitive process (Stepanov and Nyborg, 2002).…”

Section: Resultsmentioning

confidence: 88%

Cellular encoding of Cy dyes for single-molecule imaging

Leisle

Chadda

Lueck

et al. 2016

eLife

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A general method is described for the site-specific genetic encoding of cyanine dyes as non-canonical amino acids (Cy-ncAAs) into proteins. The approach relies on an improved technique for nonsense suppression with in vitro misacylated orthogonal tRNA. The data show that Cy-ncAAs (based on Cy3 and Cy5) are tolerated by the eukaryotic ribosome in cell-free and whole-cell environments and can be incorporated into soluble and membrane proteins. In the context of the Xenopus laevis oocyte expression system, this technique yields ion channels with encoded Cy-ncAAs that are trafficked to the plasma membrane where they display robust function and distinct fluorescent signals as detected by TIRF microscopy. This is the first demonstration of an encoded cyanine dye as a ncAA in a eukaryotic expression system and opens the door for the analysis of proteins with single-molecule resolution in a cellular environment.DOI: http://dx.doi.org/10.7554/eLife.19088.001

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Section: Resultsmentioning

confidence: 88%

Cellular encoding of Cy dyes for single-molecule imaging

Leisle

Chadda

Lueck

et al. 2016

eLife

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“…The Ind amino acid has seen limited use in structure-function studies for hydrogen bond testing (Lacroix et al, 2012; Pless et al, 2013, 2014; Kim et al, 2015; Zhang et al, 2015) and none thus far in structural or computational studies that would inform on its tolerance once encoded within a protein. For instance, it is not known if the Ind substitution would specifically limit hydrogen bonding at the indole nitrogen atom (the purpose for which it was designed) or could it also produce a new side-chain orientation that itself alters protein function.…”

Section: Resultsmentioning

confidence: 99%

Atomic mutagenesis in ion channels with engineered stoichiometry

Lueck

Mackey

Infield

et al. 2016

eLife

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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.DOI: http://dx.doi.org/10.7554/eLife.18976.001

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“…A conserved phenylalanine residue in the hydrophobic constriction site and the intracellular negative charge cluster has been implicated as a “gating charge transfer center (CTC)” in K V 139 channels . In Na V 1.4, these same residues only appear to be essential for proper VSD folding and membrane trafficking of the channel 140 . Nevertheless, all VSDs form an hourglass-shaped structure in the membrane that essentially functions as a voltage-dependent arginine side-chain transporter.…”

Section: Introductionmentioning

confidence: 99%

Bacterial Voltage-Gated Sodium Channels (BacNaVs) from the Soil, Sea, and Salt Lakes Enlighten Molecular Mechanisms of Electrical Signaling and Pharmacology in the Brain and Heart

Payandeh¹,

Minor

2015

Journal of Molecular Biology

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Voltage-gated sodium channels (NaVs) provide the initial electrical signal that drives action potential generation in many excitable cells of the brain, heart, and nervous system. For more than 60 years, functional studies of NaVs have occupied a central place in physiological and biophysical investigation of the molecular basis of excitability. Recently, structural studies of members of a large family of bacterial voltage-gated sodium channels (BacNaVs) prevalent in soil, marine, and salt lake environments that bear many of the core features of eukaryotic NaVs have reframed ideas for voltage-gated channel function, ion selectivity, and pharmacology. Here, we analyze the recent advances, unanswered questions, and potential of BacNaVs as templates for drug development efforts.

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Asymmetric functional contributions of acidic and aromatic side chains in sodium channel voltage-sensor domains

Abstract: Conserved acidic and aromatic residues in the four sodium channel voltage-sensor domains make domain-specific functional contributions.

Cited by 42 publications

References 60 publications

Cellular encoding of Cy dyes for single-molecule imaging

Cellular encoding of Cy dyes for single-molecule imaging

Atomic mutagenesis in ion channels with engineered stoichiometry

Bacterial Voltage-Gated Sodium Channels (BacNaVs) from the Soil, Sea, and Salt Lakes Enlighten Molecular Mechanisms of Electrical Signaling and Pharmacology in the Brain and Heart

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