Genetic Code Expansion for Mechanistic Studies in Ion Channels: An (Un)natural Union of Chemistry and Biology.

Ion channels play central roles in biology and human health by catalyzing the transmembrane flow of electrical charge. These proteins are ideal targets for genetic code expansion (GCE) methods because it is feasible to measure ion channel activity from miniscule amounts of protein and to analyze the resulting data via rigorous, established biophysical methods. In an ideal scenario, the encoding of synthetic, noncanonical amino acids via GCE allows the experimenter to ask questions inaccessible to traditional methods. For this reason, GCE has been successfully applied to a variety of ligand- and voltage-gated channels wherein extensive structural, functional, and pharmacological data exist. Here, we provide a comprehensive summary of GCE as applied to ion channels. We begin with an overview of the methods used to encode noncanonical amino acids in channels and then describe mechanistic studies wherein GCE was used for photochemistry (cross-linking; caged amino acids) and atomic mutagenesis (isosteric manipulation of charge and aromaticity; backbone mutation). Lastly, we cover recent advances in the encoding of fluorescent amino acids for the real-time study of protein conformational dynamics.

Expression and purification of fluorinated proteins from mammalian suspension culture.

The site-specific encoding of noncanonical amino acids allows for the introduction of rationalized chemistry into a target protein. Of the methods that enable this technology, evolved tRNA and synthetase pairs offer the potential for expanded protein production and purification. Such an approach combines the versatility of solid-phase peptide synthesis with the scalable features of recombinant protein production. We describe the large scale production and purification of eukaryotic proteins bearing fluorinated phenylalanine in mammalian suspension cell preparations. Downstream applications of this approach include scalable recombinant protein preparation for ligand binding assays with small molecules and ligands, protein structure determination, and protein stability assays.

Real-time observation of functional specialization among phosphorylation sites in CFTR.

Phosphoregulation is ubiquitous in biology. Defining the functional roles of individual phosphorylation sites within a multivalent system remains particularly challenging. We have therefore applied a chemical biology approach to light-control the state of single candidate phosphoserines in the canonical anion channel CFTR while simultaneously measuring channel activity. The data show striking non-equivalency among protein kinase A consensus sites, which vary from <10% to >1,000% changes in channel activity upon phosphorylation. Of note, slow phosphorylation of S813 suggests that this site is rate-limiting to the full activation of CFTR. Further, this approach reveals an unexpected coupling between the phosphorylation of S813 and a nearby site, S795. Overall, these data establish an experimental route to understanding roles of specific phosphoserines within complex phosphoregulatory domains. This strategy may be employed in the study of phosphoregulation of other eukaryotic proteins.

Tuning phenylalanine fluorination to assess aromatic contributions to protein function and stability in cells.

The aromatic side-chains of phenylalanine, tyrosine, and tryptophan interact with their environments via both hydrophobic and electrostatic interactions. Determining the extent to which these contribute to protein function and stability is not possible with conventional mutagenesis. Serial fluorination of a given aromatic is a validated method in vitro and in silico to specifically alter electrostatic characteristics, but this approach is restricted to a select few experimental systems. Here, we report a group of pyrrolysine-based aminoacyl-tRNA synthetase/tRNA pairs (tRNA/RS pairs) that enable the site-specific encoding of a varied spectrum of fluorinated phenylalanine amino acids in E. coli and mammalian (HEK 293T) cells. By allowing the cross-kingdom expression of proteins bearing these unnatural amino acids at biochemical scale, these tools may potentially enable the study of biological mechanisms which utilize aromatic interactions in structural and cellular contexts.

Role of a conserved ion-binding site tyrosine in ion selectivity of the Na+/K+ pump.

The essential transmembrane Na+ and K+ gradients in animal cells are established by the Na+/K+ pump, a P-type ATPase that exports three Na+ and imports two K+ per ATP hydrolyzed. The mechanism by which the Na+/K+ pump distinguishes between Na+ and K+ at the two membrane sides is poorly understood. Crystal structures identify two sites (sites I and II) that bind Na+ or K+ and a third (site III) specific for Na+. The side chain of a conserved tyrosine at site III of the catalytic α-subunit (Xenopus-α1 Y780) has been proposed to contribute to Na+ binding by cation-π interaction. We substituted Y780 with natural and unnatural amino acids, expressed the mutants in Xenopus oocytes and COS-1 cells, and used electrophysiology and biochemistry to evaluate their function. Substitutions disrupting H-bonds impaired Na+ interaction, while Y780Q strengthened it, likely by H-bond formation. Utilizing the non-sense suppression method previously used to incorporate unnatural derivatives in ion channels, we were able to analyze Na+/K+ pumps with fluorinated tyrosine or phenylalanine derivatives inserted at position 780 to diminish cation-π interaction strength. In line with the results of the analysis of mutants with natural amino acid substitutions, the results with the fluorinated derivatives indicate that Na+-π interaction with the phenol ring at position 780 contributes minimally, if at all, to the binding of Na+. All Y780 substitutions decreased K+ apparent affinity, highlighting that a state-dependent H-bond network is essential for the selectivity switch at sites I and II when the pump changes conformational state.

The molecular evolution of function in the CFTR chloride channel.

The ATP-binding cassette (ABC) transporter superfamily includes many proteins of clinical relevance, with genes expressed in all domains of life. Although most members use the energy of ATP binding and hydrolysis to accomplish the active import or export of various substrates across membranes, the cystic fibrosis transmembrane conductance regulator (CFTR) is the only known animal ABC transporter that functions primarily as an ion channel. Defects in CFTR, which is closely related to ABCC subfamily members that bear function as bona fide transporters, underlie the lethal genetic disease cystic fibrosis. This article seeks to integrate structural, functional, and genomic data to begin to answer the critical question of how the function of CFTR evolved to exhibit regulated channel activity. We highlight several examples wherein preexisting features in ABCC transporters were functionally leveraged as is, or altered by molecular evolution, to ultimately support channel function. This includes features that may underlie (1) construction of an anionic channel pore from an anionic substrate transport pathway, (2) establishment and tuning of phosphoregulation, and (3) optimization of channel function by specialized ligand-channel interactions. We also discuss how divergence and conservation may help elucidate the pharmacology of important CFTR modulators.

Selection and validation of orthogonal tRNA/synthetase pairs for the encoding of unnatural amino acids across kingdoms.

As an increasing number of protein structures are resolved at atomic and near-atomic resolution, conventional amino acid mutagenesis may be insufficient to test many mechanistic hypotheses. As a result, the development of new tRNA/aminoacyl-tRNA synthetase (aaRS) pairs has become an important tool for determining intricate molecular interactions and understanding protein structures. This chapter describes in detail the directed evolution of new tRNA/aaRS pairs in Escherichia coli for the incorporation of non-canonical amino acids (ncAA). Section 1 describes the selection of new tRNA/aaRS pairs in E. coli. Section 2 details the use of a synthetase to incorporate an ncAA into a mammalian cell line, and Sections 1 and 2 both include methods on the determination of synthetase efficacy and fidelity.

Cation-π Interactions and their Functional Roles in Membrane Proteins.

Cation-π interactions arise as a result of strong attractive forces between positively charged entities and the π-electron cloud of aromatic groups. The physicochemical characteristics of cation-π interactions are particularly well-suited to the dual hydrophobic/hydrophilic environment of membrane proteins. As high-resolution structural data of membrane proteins bring molecular features into increasingly sharper view, cation-π interactions are gaining traction as essential contributors to membrane protein chemistry, function, and pharmacology. Here we review the physicochemical properties of cation-π interactions and present several prominent examples which demonstrate significant roles for this specialized biological chemistry.

What biologists want from their chloride reporters - a conversation between chemists and biologists.

Impaired chloride transport affects diverse processes ranging from neuron excitability to water secretion, which underlie epilepsy and cystic fibrosis, respectively. The ability to image chloride fluxes with fluorescent probes has been essential for the investigation of the roles of chloride channels and transporters in health and disease. Therefore, developing effective fluorescent chloride reporters is critical to characterizing chloride transporters and discovering new ones. However, each chloride channel or transporter has a unique functional context that demands a suite of chloride probes with appropriate sensing characteristics. This Review seeks to juxtapose the biology of chloride transport with the chemistries underlying chloride sensors by exploring the various biological roles of chloride and highlighting the insights delivered by studies using chloride reporters. We then delineate the evolution of small-molecule sensors and genetically encoded chloride reporters. Finally, we analyze discussions with chloride biologists to identify the advantages and limitations of sensors in each biological context, as well as to recognize the key design challenges that must be overcome for developing the next generation of chloride sensors.

An Ancient CFTR Ortholog Informs Molecular Evolution in ABC Transporters.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel central to the development of secretory diarrhea and cystic fibrosis. The oldest CFTR ortholog identified is from dogfish shark, which retains similar structural and functional characteristics to the mammalian protein, thereby highlighting CFTR's critical role in regulating epithelial ion transport in vertebrates. However, the identification of an early CFTR ortholog with altered structure or function would provide critical insight into the evolution of epithelial anion transport. Here, we describe the earliest known CFTR, expressed in sea lamprey (Petromyzon marinus), with unique structural features, altered kinetics of activation and sensitivity to inhibition, and altered single-channel conductance compared to human CFTR. Our data provide the earliest evolutionary evidence of CFTR, offering insight regarding changes in gene and protein structure that underpin evolution from transporter to anion channel. Importantly, these data provide a unique platform to enhance our understanding of vertebrate phylogeny over a critical period of evolutionary expansion.

ATP-Dependent Signaling in Simulations of a Revised Model of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily that has uniquely evolved to function as a chloride channel. It binds and hydrolyzes ATP at its nucleotide binding domains to form a pore providing a diffusive pathway within its transmembrane domains. CFTR is the only known protein from the ABC superfamily with channel activity, and its dysfunction causes the disease cystic fibrosis. While much is known about the functional aspects of CFTR, significant gaps remain, such as the structure-function relationship underlying signaling of ATP binding. In the present work, we refined an existing homology model using an intermediate-resolution (9 Å) published cryo-electron microscopy map. The newly derived models have been simulated in equilibrium molecular dynamics simulations for a total of 2.5 µs in multiple ATP-occupancy states. Putative conformational movements connecting ATP binding with pore formation are elucidated and quantified. Additionally, new interdomain interactions between E543, K968, and K1292 have been identified and confirmed experimentally; these interactions may be relevant for signaling ATP binding and hydrolysis to the transmembrane domains and induction of pore opening.

Engineered transfer RNAs for suppression of premature termination codons.

Premature termination codons (PTCs) are responsible for 10-15% of all inherited disease. PTC suppression during translation offers a promising approach to treat a variety of genetic disorders, yet small molecules that promote PTC read-through have yielded mixed performance in clinical trials. Here we present a high-throughput, cell-based assay to identify anticodon engineered transfer RNAs (ACE-tRNA) which can effectively suppress in-frame PTCs and faithfully encode their cognate amino acid. In total, we identify ACE-tRNA with a high degree of suppression activity targeting the most common human disease-causing nonsense codons. Genome-wide transcriptome ribosome profiling of cells expressing ACE-tRNA at levels which repair PTC indicate that there are limited interactions with translation termination codons. These ACE-tRNAs display high suppression potency in mammalian cells, Xenopus oocytes and mice in vivo, producing PTC repair in multiple genes, including disease causing mutations within cystic fibrosis transmembrane conductance regulator (CFTR).

Structural basis of α-scorpion toxin action on Nav channels.

Fast inactivation of voltage-gated sodium (Nav) channels is essential for electrical signaling, but its mechanism remains poorly understood. Here we determined the structures of a eukaryotic Nav channel alone and in complex with a lethal α-scorpion toxin, AaH2, by electron microscopy, both at 3.5-angstrom resolution. AaH2 wedges into voltage-sensing domain IV (VSD4) to impede fast activation by trapping a deactivated state in which gating charge interactions bridge to the acidic intracellular carboxyl-terminal domain. In the absence of AaH2, the S4 helix of VSD4 undergoes a ~13-angstrom translation to unlatch the intracellular fast-inactivation gating machinery. Highlighting the polypharmacology of α-scorpion toxins, AaH2 also targets an unanticipated receptor site on VSD1 and a pore glycan adjacent to VSD4. Overall, this work provides key insights into fast inactivation, electromechanical coupling, and pathogenic mutations in Nav channels.

Main-chain mutagenesis reveals intrahelical coupling in an ion channel voltage-sensor.

Membrane proteins are universal signal decoders. The helical transmembrane segments of these proteins play central roles in sensory transduction, yet the mechanistic contributions of secondary structure remain unresolved. To investigate the role of main-chain hydrogen bonding on transmembrane function, we encoded amide-to-ester substitutions at sites throughout the S4 voltage-sensing segment of Shaker potassium channels, a region that undergoes rapid, voltage-driven movement during channel gating. Functional measurements of ester-harboring channels highlight a transitional region between α-helical and 310 segments where hydrogen bond removal is particularly disruptive to voltage-gating. Simulations of an active voltage sensor reveal that this region features a dynamic hydrogen bonding pattern and that its helical structure is reliant upon amide support. Overall, the data highlight the specialized role of main-chain chemistry in the mechanism of voltage-sensing; other catalytic transmembrane segments may enlist similar strategies in signal transduction mechanisms.

Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape.

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.

Orthogonality of Pyrrolysine tRNA in the Xenopus oocyte.

Chemical aminoacylation of orthogonal tRNA allows for the genetic encoding of a wide range of synthetic amino acids without the need to evolve specific aminoacyl-tRNA synthetases. This method, when paired with protein expression in the Xenopus laevis oocyte expression system, can extract atomic scale functional data from a protein structure to advance the study of membrane proteins. The utility of the method depends on the orthogonality of the tRNA species used to deliver the amino acid. Here, we report that the pyrrolysyl tRNA (pylT) from Methanosarcina barkeri fusaro is orthogonal and highly competent for genetic code expansion experiments in the Xenopus oocyte. The data show that pylT is amendable to chemical acylation in vitro; it is then used to rescue a cytoplasmic site within a voltage-gated sodium channel. Further, the high fidelity of the pylT is demonstrated via encoding of lysine within the selectivity filter of the sodium channel, where sodium ion recognition by the distal amine of this side-chain is essential. Thus, pylT is an appropriate tRNA species for delivery of amino acids via nonsense suppression in the Xenopus oocyte. It may prove useful in experimental contexts wherein reacylation of suppressor tRNAs have been observed.

Rapid evolution of a voltage-gated sodium channel gene in a lineage of electric fish leads to a persistent sodium current.

Most weakly electric fish navigate and communicate by sensing electric signals generated by their muscle-derived electric organs. Adults of one lineage (Apteronotidae), which discharge their electric organs in excess of 1 kHz, instead have an electric organ derived from the axons of specialized spinal neurons (electromotorneurons [EMNs]). EMNs fire spontaneously and are the fastest-firing neurons known. This biophysically extreme phenotype depends upon a persistent sodium current, the molecular underpinnings of which remain unknown. We show that a skeletal muscle-specific sodium channel gene duplicated in this lineage and, within approximately 2 million years, began expressing in the spinal cord, a novel site of expression for this isoform. Concurrently, amino acid replacements that cause a persistent sodium current accumulated in the regions of the channel underlying inactivation. Therefore, a novel adaptation allowing extreme neuronal firing arose from the duplication, change in expression, and rapid sequence evolution of a muscle-expressing sodium channel gene.

Insulin signaling via the PI3-kinase/Akt pathway regulates airway glucose uptake and barrier function in a CFTR-dependent manner.

Cystic fibrosis-related diabetes is the most common comorbidity associated with cystic fibrosis (CF) and correlates with increased rates of lung function decline. Because glucose is a nutrient present in the airways of patients with bacterial airway infections and because insulin controls glucose metabolism, the effect of insulin on CF airway epithelia was investigated to determine the role of insulin receptors and glucose transport in regulating glucose availability in the airway. The response to insulin by human airway epithelial cells was characterized by quantitative PCR, immunoblot, immunofluorescence, and glucose uptake assays. Phosphatidylinositol 3-kinase/protein kinase B (Akt) signaling and cystic fibrosis transmembrane conductance regulator (CFTR) activity were analyzed by pharmacological and immunoblot assays. We found that normal human primary airway epithelial cells expressed glucose transporter 4 and that application of insulin stimulated cytochalasin B-inhibitable glucose uptake, consistent with a requirement for glucose transporter translocation. Application of insulin to normal primary human airway epithelial cells promoted airway barrier function as demonstrated by increased transepithelial electrical resistance and decreased paracellular flux of small molecules. This provides the first demonstration that airway cells express insulin-regulated glucose transporters that act in concert with tight junctions to form an airway glucose barrier. However, insulin failed to increase glucose uptake or decrease paracellular flux of small molecules in human airway epithelia expressing F508del-CFTR. Insulin stimulation of Akt1 and Akt2 signaling in CF airway cells was diminished compared with that observed in airway cells expressing wild-type CFTR. These results indicate that the airway glucose barrier is regulated by insulin and is dysfunctional in CF.

Cellular encoding of Cy dyes for single-molecule imaging.

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.

Atomic mutagenesis in ion channels with engineered stoichiometry.

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.