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. 2015 Mar 13;593(Pt 12):2627–2634. doi: 10.1113/jphysiol.2014.287714

Atom-by-atom engineering of voltage-gated ion channels: Magnified insights into function and pharmacology

Stephan A Pless 1, Robin Y Kim 2, Christopher A Ahern 3,✉, Harley T Kurata 2
PMCID: PMC4500348  PMID: 25640301

Abstract

Unnatural amino acid incorporation into ion channels has proven to be a valuable approach to interrogate detailed hypotheses arising from atomic resolution structures. In this short review, we provide a brief overview of some of the basic principles and methods for incorporation of unnatural amino acids into proteins. We also review insights into the function and pharmacology of voltage-gated ion channels that have emerged from unnatural amino acid mutagenesis approaches.

Introduction

Ion channel proteins are fundamental effectors of electrical excitability and rapid signalling in the body. Consequently, they are a very important class of drug targets, and are also recognized to underlie undesired side-effects of many drugs. Significant progress over the past decade in membrane protein structural biology has advanced our understanding of ion channels, first with several breakthrough K+ channel structures (Doyle et al. 1998; Zhou et al. 2001), followed by a steady emergence of structures of other channel types (Dutzler et al. 2002; Jiang et al. 2003; Long et al. 2005; Nishida et al. 2007; Payandeh et al. 2011). This revolution in our structural understanding of ion channels has shifted experimental approaches, and has provided new challenges related to the specificity and detail with which we unravel fundamental elements of ion channel function.

Since the first ion channels were cloned, function and pharmacology of ion channels has been primarily investigated with mutagenesis approaches, in which the effects of mutations were used to divine sites for drug or ligand binding, structural details of conformational changes, and other questions of fundamental importance. However, these alterations, and the insights they provide, are generally ‘coarse-grained’. While conventional site-directed mutagenesis may inform us whether a particular amino acid is involved in gating or drug binding, it generally does not enable more detailed questions about these processes. With the advent of atomic resolution structures and ever-growing mechanistic understanding of ion channels, this shortcoming becomes limiting, because we are now able to rationalize structure-based hypotheses related to specific chemical forces that underlie drug binding or channel function. In this short review we hope to introduce approaches for incorporation of unnatural amino acids (UAAs) that can be used to evaluate these detailed questions. We will also review some recent insights into function and pharmacology of voltage-gated ion channels that have emerged from these approaches.

UAA mutagenesis principles and methodology

The expanding body of structural data for ion channel proteins argues for the increased application of methods for atom-by-atom engineering of protein structure. Methods for UAA mutagenesis are currently the most practical approaches for this level of precision in unravelling functional principles suggested by atomic resolution structures. Until recently, the finely detailed manipulations enabled by UAA mutagenesis had been applied to ion channel studies primarily involving Cys-loop receptors like the nicotinic and ionotropic GABA receptors (Li et al. 2001; Beene et al. 2004; Lummis et al. 2005; Blum et al. 2011). More recently, these approaches have developed more universal appeal and found applications in several voltage-gated ion channel types (discussed later).

The most common approach for introduction of UAAs is by ‘nonsense suppression’ of a stop codon (usually a TAG ‘amber’ stop codon) introduced into the coding sequence of a gene of interest (Fig.1). The core principle of nonsense suppression methods is the generation of an unnatural tRNA acylated with the desired UAA, which is able to direct amino acid incorporation at the introduced stop codon, and thereby overcome the translation termination machinery that would normally intervene. There are numerous reviews and primary literature describing the fine details of these methods (Beene et al. 2003; Davis & Chin, 2012), so we will provide a very brief overview of the main approaches for nonsense suppression in voltage-gated ion channels, and other methods for UAA mutagenesis:

Figure 1.

Figure 1

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Basic methods for UAA mutagenesis by nonsense suppression

The basic principle for nonsense suppression is to introduce an orthogonal tRNA loaded with the UAA, that can direct its incorporation into a nascent polypeptide (grey squares) at the site of a stop codon (usually UAG) that has been introduced into a gene of interest. Generation of this tRNA can be done synthetically (top left panel) by ligating a UAA with a synthetic tRNA in vitro, followed by injection of the product (aminoacylated-tRNA) into an expression system along with mRNA for the gene of interest (Dougherty & Van Arnam, 2014). Cells can also be engineered to generate the required tRNA in vivo by introducing plasmids to drive expression of an orthogonal tRNA: aminoacyl-tRNA synthetase pair (Davis & Chin, 2012). The engineered synthetase must be able to recognize the foreign tRNA and the desired UAA (usually added to cellular growth media), and catalyse their ligation to generate an aminoacylated-tRNA that can be incorporated into the gene of interest.

In vitro synthesis of amino-acylated tRNA and microinjection into Xenopus laevis oocytes

With this approach, tRNA and UAAs are synthesized and ligated in vitro (Fig.1, left panel), and introduced into cells (for the study of ion channels, this is typically achieved via microinjection into Xenopus laevis oocytes; Noren et al. 1989; Nowak et al. 1998). A chemical architecture has been developed and optimized that allows a wide variety of amino acids to be ligated and delivered via a standard tRNA template. This flexibility has several significant benefits (Dougherty & Van Arnam, 2014). Firstly, the nature of the in vitro synthesis allows for very finely altered amino acids to be introduced (that may be difficult to engineer directed evolution of a synthetase; see next section). This has been particularly beneficial in our investigation of sites that are highly sensitive and only tolerate subtle structural changes (section headed ‘Enigmatic aromatics and other tales from the front line’). Amino acids with exotic functionality or structure can also be introduced by the same method. This method can be implemented with relatively small scale chemical syntheses, allowing for efficient channeling of resources in an academic lab, and rapid introduction of newly synthesized amino acids into the workflow. Importantly, Xenopus oocytes are a commonly used ‘workhorse’ expression system for electrophysiology, and so the adaptation of nonsense suppression methods for this recording system has been ideal for the application of UAA mutagenesis approaches in electrophysiology (and is the approach used most commonly by our research groups).

Directed evolution of amino-acyl tRNA synthetases for expression in mammalian cell lines

This approach was first pioneered for incorporation of UAAs in prokaryotic cells, and has since been modified to enable UAA incorporation in mammalian cell lines, primary neuronal cultures, and even model organisms (Xie & Schultz, 2005; Wang et al. 2007b; Kang et al. 2013; Elliott et al. 2014). This method relies on the expression of tRNA: aminoacyl-tRNA synthetase pairs in cells, that are bio-orthogonal to the native protein translation system (i.e. native synthetase enzymes will not recognize the foreign tRNA, and the foreign tRNA synthetase will not recognize native tRNAs or amino acids, Fig.1, right panel; Davis & Chin, 2012). The primary hurdle for this approach is the generation of engineered amino-acyl tRNA synthetases that no longer recognize their naturally-occurring amino acid substrate (in favour of a particular UAA). This step can be time consuming, but once complete enables repeated use of the engineered synthetase by transfection into cells, and generates a highly portable tool that can be shared between research groups. However, one potential drawback of this approach is that it may be difficult to engineer synthetases that can discriminate subtly altered UAAs from the native substrate of the enzyme. Nevertheless, this approach is developing rapidly to include a broader number of synthetase–tRNA combinations (Chin & Schultz, 2002; Ye et al. 2008; Lang et al. 2012), modifications of the method to exploit quadruplet stop codon recognition (Neumann et al. 2010), and alterations to ribosome components and termination release factor (eRF1) to enable more efficient expression of engineered proteins (Wang et al. 2007a; Schmied et al. 2014). This method has not been as widely used for ion channel studies relative to the in vitro synthesis approach (Wang et al. 2007b; Kang et al. 2013; Klippenstein et al. 2014), but the convenience and accelerating development of these tools suggest that their application will rapidly become more commonplace.

Merging approaches for novel applications

Recently, the previously clear distinction between the approaches mentioned in the previous sections have become more blurry, as the use of the engineered tRNA–synthetase pairs has been pioneered for use in Xenopus laevis oocytes (Kalstrup & Blunck, 2013; Zhu et al. 2014). Similarly, it is not inconceivable that in vitro amino acylated tRNA could be injected into mammalian cells in the future.

Semi-synthetic ion channel generation

Lastly, a noteworthy but less frequently employed approach for introduction of UAAs into ion channels has been by ‘semi-synthesis’, in which multiple fragments of an ion channel are synthesized in recombinant systems (Muralidharan & Muir, 2006). This allows for a chemical approach to add a non-native amino acid to one or both of the synthesized fragments, followed by ligation of the fragments to form a full-length ion channel protein. The application of semi-synthesis to ion channel studies has been notably used for the incorporation of amino acids with stereochemistry that would not be tolerated by the ribosomal translation system (Valiyaveetil et al. 2004). Protein manufactured with this approach has been used for functional recordings of ion channels (KcsA and KvAP), and in some cases has been used to generate large enough protein quantities for crystallographic studies (Valiyaveetil et al. 2006; Devaraneni et al. 2013; Matulef et al. 2013).

Where things can go wrong – checks and balances in the generation of engineered ion channel proteins

Anecdotally, not all ion channels, and not all residues within a particular ion channel, are amenable to these approaches. A common pitfall with stop codon suppression approaches is that the protein translation machinery may incorporate undesired amino acids at the location of the introduced stop codon (sometimes referred to as ‘bleedthrough’). As an important precaution, a ‘subtraction of parts’ approach is commonly used for control in these experiments, in which a component is omitted from the system (for example, by injecting Xenopus oocytes with a ‘cargo-less’ tRNA). Ideally, this will result in the absence of functional ion channel expression, although occasionally we have observed the emergence of currents indicating that the expression system has managed to overcome the stop signal, and introduce an undesired amino acid (Pless et al. 2014). At present, this phenomenon is difficult to predict, although hopefully with ongoing application of these approaches, a better understanding of the contextual features that allow for ‘readthrough’ of the stop codon will emerge. A second shortcoming that is frequently encountered is that nonsense suppression methods inherently generate a substantial amount of truncated protein, due to inefficient readthrough of the introduced stop codon. This reduces overall expression levels of the protein of interest, and in some cases the truncated subunits may exert a dominant negative effect that suppresses function of the desirably engineered protein (Ahern & Kobertz, 2009). Recent reports of more efficient incorporation of UAAs after co-expression of a mutated version of eRF1 may help to overcome this challenge (Schmied et al. 2014).

Enigmatic aromatics and other tales from the front line – case studies in the application of UAAs to understand voltage-gated ion channel function and pharmacology

In the context of voltage-gated ion channel function and pharmacology, UAA mutagenesis has been applied most advantageously in situations where conventional mutagenesis is highly perturbative (i.e. to enable subtle alterations of side-chains that are extremely intolerant to conventional mutations), or to distinguish subtle chemical details that cannot be tested with conventional methods. The aromatics (Phe, Trp, Tyr) are a useful example to discuss the benefits of UAA mutagenesis, because of the diverse chemical forces they are able to deploy to interact with ligands, and because of the lack of naturally occurring amino acids that specifically modify these features (Beene et al. 2003; Santarelli et al. 2007; Ahern et al. 2008). Moreover, aromatic side-chains are recognized to make important contributions to structural motifs, domain interfaces, and ligand binding sites that strongly regulate ion channel function (West et al. 1992; Ragsdale et al. 1996; Doyle et al. 1998; Tao et al. 2010). A frequent oversimplification when describing aromatics (often in the context of drug binding) is to refer to them as hydrophobic. For example, it is not uncommon to come across the term ‘hydrophobic binding pocket’ in reference to drug binding sites comprising one or more aromatic side-chains. However, their complex electronic arrangement, and propensity to form hydrogen bonds and cation–π interactions can generate subtle interactions that accommodate small polar ligands like nicotine, neurotransmitters, certain local anaesthestics, and others (Ahern et al. 2008; Xiu et al. 2009).

UAA mutagenesis is an ideal approach to deconstruct the complex interactions formed by aromatic side-chains, enabling subtle alterations in the charge distribution (using fluorinated analogues), or altered hydrogen bonding properties, while only minimally changing the shape, size, hydrophobicity, or other properties of the side-chain. These methods are of course also applicable to subtle derivatives of other side-chains, and for the introduction of more exotic amino acids with unique properties (e.g. fluorescence, cross-linking). We provide a brief overview of several recent examples of the application of these tools in voltage-gated ion channels to highlight the value of this experimental approach.

Atomic level insights into regulation of K+ channel activation

Activation of Kv channels requires a coordinated interplay of numerous conformational rearrangements in response to a change in membrane potential. This process is initiated by the so-called voltage-sensing domains (VSDs), which are modular domains that are physically linked to the central ion-conducting pore domain (PD). The actual voltage-sensor in the VSD is an α-helical segment (S4) containing a number of positively charged amino acids (Fig.2). Based on results from conventional mutagenesis and structural data, these were thought to interact with an intracellular (Shaker residues Glu293, Asp316) and an extracellular cluster (Glu283) of negatively charged amino acids (INC and ENC, respectively; Papazian et al. 1995; Long et al. 2007), as well as a highly conserved aromatic (Phe290) sometimes referred to as the ‘gating charge transfer centre’ (Tao et al. 2010). Unique experimental outcomes in the INC/ENC highlight that a conventional mutagenesis strategy of replacing Asp/Glu side-chains with Asn/Gln can, in certain cases, be deceptive: this not only neutralizes the side-chain in question, but also replaces a potent H-bond acceptor group with a strong H-bond donor moiety. Moreover, these conventional substitutions were extremely disruptive to channel gating and trafficking (Papazian et al. 1995). Instead, using more subtle, neutral analogues of Asp/Glu that cannot act as H-bond donors (Fig.2), it was demonstrated that only the ENC, and not the INC, forms energetically significant interactions with S4 in both Kv and Nav channels (Pless et al. 2011b, 2014). UAA mutagenesis was also applied to understand the molecular details of interactions between Phe290 and S4 gating charges, demonstrating that a cation–π interaction did not make significant contributions to gating, but could be introduced by a Phe290Trp mutation (Tao et al. 2010; Pless et al. 2011b). Again, unnatural substitutions of Phe and Trp analogues at Phe290 were a valuable experimental approach, because conventional mutagenesis at this site tended to be extremely disruptive to channel function.

Figure 2.

Figure 2

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UAA alternatives to investigate acidic side-chains

UAA mutagenesis has been used to investigate interactions between charged and aromatic residues in the K+ channel voltage-sensing domain, shown on the left. Voltage-sensing Arg and Lys residues are highlighted in red, while essential acidic (Glu 283, Glu 293, Asp 316) and aromatic residues (Phe 290) are highlighted in yellow. Structures for conventional ‘conservative’ mutations using conventional methods (Asn and Gln) are illustrated, along with some alternatives that can be incorporated via UAA mutagenesis (Nha, nitrohomoalanine; Akp, 2-amino-4-ketopentanoic acid). The UAAs have the benefit of neutralizing the charge of native acidic residues, while not introducing strong H-bond donors, leading to markedly different effects on ion channel function relative to Asn or Gln substitutions (Pless et al. 2011b).

UAA mutagenesis has also provided novel insights into the detailed energetic balance of open and closed states of the PD, and what detailed forces might shape an intrinsic bias of the PD towards the closed state (Yifrach & MacKinnon, 2002; Jensen et al. 2012). The underlying mechanism for this preference remained elusive, although a cluster of aromatic amino acids near the intracellular end of the PD had been suggested to play a role. Of these side-chains, Phe481 (Shaker residue numbering) is the most highly conserved, but had proven extremely challenging to study, since virtually all conventional mutations in this position result in non-functional channels. We approached this problem by introducing serially fluorinated Phe derivatives to subtly alter only the electrostatic surface potential (ESP) of Phe481, but not its size or hydrophobicity. Using these derivatives in combination with computational approaches, we generated evidence for a repulsive open-state interaction between the negative ESP of Phe481 and a nearby negatively charged side-chain, Glu395 (Pless et al. 2013b). Apart from shedding new light on the role of a residue that could not be studied with conventional methods, this example also brings attention to the possibility of inherently repulsive interactions that can fine tune the equilibrium between distinct states of proteins such as ion channels.

Atomic level insights into regulation of K+ channel inactivation

After opening, many Kv channels undergo spontaneous pore closure referred to as inactivation. Inactivation is broadly discriminated into two kinetically distinct processes: fast (or N-type) inactivation is mediated by a well-characterized ‘ball-and-chain’-type mechanism (Hoshi et al. 1990); whereas slow (or C-type) inactivation is a more complex process, probably involving a number of side-chains in different channel domains (Kurata & Fedida, 2006; Cuello et al. 2010; Hoshi & Armstrong, 2013). We applied UAA mutagenesis to investigate the critical role of side-chains around the selectivity filter (SF), especially Trp434 (Shaker numbering) (Perozo et al. 1993). However, mutating Trp434 into any naturally occurring side-chain alters numerous physico-chemical properties, such as size and H-bonding ability, and ablates channel function. For this reason, we chose to probe the role of Trp434 by using unnatural Trp derivatives that either abrogate or increase its propensity to act as a H-bond donor without altering other side-chain properties (Fig.3). These experiments firmly established the role of a H-bond at the extracellular mouth of the SF that regulates entry into the inactivated state (Pless et al. 2013a). UAA mutagenesis approaches have also been used to investigate the relationship between ion permeation and C-type inactivation, by engineering ion channels with altered ion occupancy of specific binding sites in the selectivity filter (Devaraneni et al. 2013; Matulef et al. 2013).

Figure 3.

Figure 3

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Sample UAA alternatives to investigate aromatic side-chains

UAA mutagenesis was employed to investigate the role of H-bonding between Shaker residues Trp 434 and Asp 447 in the selectivity filter region. All mutations to Trp 434 dramatically perturb channel function, and there are no conventional mutagenesis options that mimic the bicyclic structure of Trp (the closest naturally-encoded side-chains are Phe and Tyr). UAA mutagenesis allows incorporation of subtly altered Trp analogues including fluorinated Trp (to alter side-chain electrostatics by manipulating the distribution of π electrons), or a useful analogue named ‘Ind’ that alters the position of the indole ring nitrogen atom (thereby abolishing the ability to H-bond). These analogues have been used to correlate Asp447:Trp434 H-bond strength with channel inactivation kinetics (Pless et al. 2013a).

Application of UAA mutagenesis to drug binding

In addition to addressing detailed questions related to protein function, UAA mutagenesis has been applied to better understand the detailed chemistry of drug binding in voltage-gated ion channels. In Nav channels, the presence of two conserved aromatics (Phe1579 and Tyr1586 in Nav1.4, Phe1760 and Trp1767 in Nav1.5, in the pore-forming S6 helix of domain IV) form an essential binding site for local anaesthetic/anti-arrhythmic drugs like lidocaine (Ragsdale et al. 1994). Careful decomposition of the forces underlying drug binding in this region have highlighted the importance of cation–π interactions for the binding of certain drug classes (notably, the empirical Vaughan-Williams class Ib anti-arrhythmics lidocaine and mexiletine; Pless et al. 2011a). The importance of this finding is that it suggests specific chemical forces that can be rationally engineered into (or out of) a drug to shape its physiological outcome. A similar scenario exists in HERG channels, in which two pore-lining aromatics (Tyr652 and Phe656) have emerged as determinants of the unusual susceptibility of HERG channels for blockade by a wide range of drugs (Mitcheson et al. 2000; Sanguinetti et al. 2005). This has become an important consideration during drug development as the cause of drug-induced long QT syndrome (Sanguinetti & Tristani-Firouzi, 2006). Conventional site-directed mutagenesis has identified these residues as important contributors to channel gating and drug binding, and offered some insights into potential mechanisms. However, the diverse potential interactions of these aromatics could be further tested using UAA mutagenesis, and might provide a valuable contribution by confirming the detailed chemical features of these side-chains that underlie their role in channel function and pharmacology. A deeper understanding of these interactions may allow for the rational development of more selective and potent drugs devoid of unwanted actions on other targets, and undesired HERG interactions to be rationally engineered out of otherwise useful drugs.

New horizons

The above examples have primarily focused on using subtle analogues of naturally occurring amino acids to study various aspects of ion channel biology. It will therefore be exciting to see how the recently established use of fluorescent UAAs for methods such as voltage-clamp fluorometry (Kalstrup & Blunck, 2013) or even single molecule fluorescence measurements (Pantoja et al. 2009) will contribute to our understanding of the dynamics of ion channel function and pharmacology.

Summary

Application of UAA mutagenesis to ion channel proteins has provided a valuable toolkit for ‘atom-by-atom engineering’ in order to investigate function with high precision. Continued development and application of these methods will help further illuminate fundamental principles of ion channel function and drug binding.

Glossary

Akp

2-amino-4-ketopentanoic acid

ENC

extracellular negatively charged (amino acids)

ESP

electrostatic surface potential

INC

intracellular negatively charged (amino acids)

Nha

nitrohomoalanine

PD

pore domain

SF

selectivity filter

UAA

unnatural amino acid

VSD

voltage-sensing domain

Biographies

Inline graphicChristopher Ahern received his BS in Chemistry and PhD in Physiology at the University of Wisconsin-Madison. He began working with genetic code expansion approaches during his post-doctoral studies with Dr Richard Horn at Jefferson Medical College through a collaboration with Drs Henry Lester and Dennis Dougherty at the California Institute of Technology. He is currently an Associate Professor in the Department of Molecular Physiology and Biophysics at the University of Iowa. For the past 5 years, the Ahern and Kurata laboratories have collaborated on the application of unnatural amino acids to the study of ion channel proteins.

Inline graphicHarley Kurata received his PhD in Physiology from the University of British Columbia studying voltage-gated potassium channels with David Fedida. He moved on to Washington University School of Medicine where he did post-doctoral research on KATP channels and in ward rectification with Colin Nichols. He is currently an Associate Professor in Pharmacology at the University of British Columbia

Additional information

Competing interests

None declared.

Funding

Work in our research groups has been supported by CIHR MOP-97988 and an HFSP Young Investigator award to H.T.K., and a CIHR CGS-M award to R.Y.K. H.T.K. is a Michael Smith Foundation for Health Research Scholar, and a CIHR New Investigator. S.A.P. is supported by a Lundbeck Foundation Fellowship. C.A.A is supported by NIH/NIGMS GM106569 and is a member of the Membrane Protein Structural Dynamics Consortium which is funded by NIH/NIGMS grant no. GM087519.

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