Site-directed mutagenesis allows one to sample the available palette of twenty amino acids. This simple exchange will simultaneously impact the chemical attributes of the side-chain (size, shape, hydrophobity, etc.) and, with luck, alter a measurable attribute of the channel in an informative way (permeability, gating, drug block). Studies adopting this approach have been successful in the identification of amino acids that contribute to gating, voltage sensing and selectivity, in addition to side-chains that engage therapeutics, blockers and toxins. Despite these significant gains, the method has its drawbacks. First, sited-directed mutagenesis rarely gives insight into the role that the original side-chain played, aside that it did contribute in some way, because it is difficult to isolate the chemical property or properties that were perturbed with mutation. This is especially true in cases where the mutation is lethal. In general, mutations exert their effects for mostly unknown reasons. Second, chemical modification of targeted amino acids, through site-directed mutation to cysteine for instance, is limited to water accessible surfaces outside of the membrane, leaving the hydrophobic nether regions of the channel uninvestigated. Third, site-directed mutagenesis is limited to amino acids available in the cell that are compatible with the ribosomal machinery, ruling out experiments that may require lines of questioning that go beyond naturally existing amino acids. As a remedy for these shortcomings, serial breakthroughs in protein chemistry have brought within the grasp of ion channel researchers methods that allow for the site-directed introduction of so-called ‘unnatural’ amino acids into a protein sequence. To date three distinct methods have been used to incorporate both subtle and extreme variants of naturally occurring side chains into K+ channels. (1) In vivo nonsense suppression in Xenopus oocytes. (2) Directionally evolved tRNA/aminoacyl-tRNA synthestase pairs that permit the study of unnatural amino acid containing proteins in mammalian cells and cultured neurons. (3) Expressed protein ligation (EPL) and protein-trans-splicing (PTS) for studying semi-synthetic channels with artificial bilayers and structural biology.
nonsense suppression allows for the site-directed incorporation of tailor-made unnatural amino acids into proteins upon the successful completion of a series of biochemical and chemical steps (46
). The first is to enlist conventional site-directed mutagenesis to introduce a TAG codon (nonsense) at the site where the unnatural amino acid will be targeted, shown as the resulting UAG on the mRNA transcript in . The second step is the construction of the tRNA loaded with the unnatural amino acid. To do this, a dinucleotide, pdCpA, is acylated with a cyanomethyl-activated, NVOC-protected unnatural amino acid, which is subsequently ligated to a truncated amber suppressor tRNA lacking its terminal two residues. A tRNA from the Tetrahymena thermophila
THG73 is used, which also carries a G73 mutation to obscure the recognition by the Xenopus
Glu amino-acyl-tRNA synthetases to prevent re-acylation of the uncharged tRNA. Generation of the acylated dinucleotide requires the synthesis of pdCpA and the NVOC-protected, activated ester of the desired unnatural amino acid. For experimentalists lacking direct access to chemical synthesis resources, several commercial operations are willing, for a price, to manufacture the appropriately derivatized amino acid and dinucleotide building blocks. With both RNA molecules in hand, the third step is co-injection into Xenopus
oocytes for expression and characterization, often by two-electrode voltage clamp. Unwanted counterfeit proteins produced by either TAG codon ‘bleed-through’ or tRNA manipulation by endogenous tRNA synthetases are controlled for by examining oocytes co-injected with the cRNA and an ‘uncharged’ THG73 tRNA that lacks an appended amino acid. This general approach has been used successfully to express numerous side-chain and back-bone mutations into a variety of ligand and voltage-gated ion channels.
Unnatural amino acid incorporation into K+ channels
The nonsense suppression method was first applied to Shaker
channels to introduce the photocleavable amino acid (2-nitrophenyl)glycine (Npg) into the cytosolic N-terminal tether that connects the inactivation ball to the channel. Photo-cleavage of the peptide backbone and the concomitant loss of the “inactivation ball” and fast inactivation demonstrate both the unnatural amino acid incorporation and the potential for the method (48
). Next, Lu and coworkers targeted ester carbonyl backbone variants of Phe and Tyr into the selectivity filter of Kir1.1 and Kir2.1 inward rectifier channels (49
). This region of the channel is especially resistant to traditional site-directed mutagenesis, as side-chain replacement strategies often fail to produce functional channels. Here the mutant channels were characterized via single channel analysis, a truly heroic undertaking, demonstrating that the peptide backbone connecting this stretch of highly conserved amino acids contributes to K+
channel gating. Lastly, the environmentally sensitive fluorescent side-chain, 6-dimethylamino-2-acyl naphthalene (AlaDan) has been incorporated into Shaker
and Kir2.1. Although K+
channels harboring this fluorescent side chain are functional, fluorescence signals from the resultant channels were not reported (50
). Each of these examples () serves to push the chemical diversity that can be introduced into K+
channels using nonsense suppression.
While nonsense suppression allows for the incorporation of a wide-variety of amino acids with structurally diverse side chains, modest side chain alterations are equally informative. Serial fluorination of aromatic phenylalanine () or tryptophan residues has been used for the investigation of putative cation-pi interactions between the candidate aromatic side-chains and cationic molecules. This manipulation titrates the negative electrostatic potential off the face of the aromatic and leaves the size, shape, polarizability and, importantly, hydrophobicity unchanged (51
). As opposed to the limited predictive capability of standard mutagenesis, here each added fluorine reduces the affinity for the ligand or blocker in a step-wise manner for a legitimate cation-pi pair. Plotting the relative lost binding energy (ΔΔG) per fluorine versus the ab initio
calculated energies for the equivalent theoretical system gives an approximation of the strength of the interaction. Using this approach, cation-pi interactions have been demonstrated to underlie ligand binding to nicotinic acetylcholine, 5-HT3 (serotonin), GABAC
and glycine receptors (52
). Energetically significant cation-pi interactions have also been shown to support the extracellular block by either tetrodotoxin (TTX) or calcium ions of voltage-gated sodium channels and their inhibition by local anesthetics (58
). Lastly, such interactions underlie the external tetraethyl ammonium (TEA) blockade of Shaker
channels where four aromatic phenylalanine residues near the selectivity filter coordinate a single blocking molecule (61
). Moreover, the cation-pi interaction is geometrically constrained such that the blocker must interact with face, not the edge, of the aromatic. Thus, evidence of a cation-pi interaction provides direct information on side-chain orientation. In addition to the blockers, drugs and ligands noted here, cation-pi interactions may also play a role in the structure-function relationships throughout biology. A theoretical study of high resolution PDB files with a data set of over 200,000 side-chains concluded that roughly 1 in every
77 residues in any given protein has a energetically significant cation-pi interaction, roughly half the incidence of predicted salt-bridges (62
). The role, if any, of putative cation-pi interactions in Shaker
channels for voltage-sensing or gating mechanisms has yet to be empirically determined, but the tools to do so are within reach.
The current iteration of the in vivo
nonsense suppression method is streamlined for the study of proteins in the Xenopus
oocyte. Therefore the recent demonstration of a suppressed UAG codon in a green fluorescent protein (GFP) expressed in cultured neurons expands the potential for more sophisticated study of the biology of ion channels in the context of a mammalian cell (63
). The method, advanced by the Schultz laboratory, employs co-evolved orthogonal tRNA/amnioacyl-tRNA synthetase pairs from the archea Methanococcus jannaschii,
(recently reviewed in (64
)). This approach (), like in vivo
nonsense suppression, uses a UAG nonsense mutation in the cRNA channel transcript to guide the tRNA to the site of incorporation, but instead of chemically pre-acylating the tRNA in vitro
, a co-expressed tRNA synthetase serves to append the tRNA with the unnatural cargo. The method relies on a specialized tRNA synthetase that has been evolved to recognize the appropriate tRNA. As with nonsense suppression, the tRNA-synthetase pair should be unrecognized by the host cell so that the tRNA is not mis-labeled or the tRNA synthetase labels endogenous tRNA molecules inappropriately. Unlike nonsense suppression, the tRNA synthetase continues to recycle (and recharge) the tRNA molecule and therefore, in theory, produces a higher density of mutant channels. Once a pair is developed for a particular unnatural amino acid, the plasmids can be readily shared, reducing the synthetic chemistry component of the approach. But because the tRNA synthetase continuously re-acylates the tRNA, the unnatural amino acid has to readily enter the cell and be present at millimolar levels.
This method has been used to incorporate a number of side-chains and those with fluorescent (65
) or chemically reactive characteristics (66
) may be of special interest for structure-function studies in ion channels. For K+
channels, tRNA-synthetase pairs have been used to target side-chains of increasing volume in the N-terminal inactivation particle of Kv1.4 channels (63
). In this particular study, each side-chain variant produced a high density of potassium current with gating behavior similar to wild-type channels except that larger side-chains inactivate on a slower time course. This difference was attributed to the steric effects of bigger side-chains having trouble accessing their binding site in the inner vestibule (). Although these experiments provide an insightful proof of principal, the true potential for this approach lies in its application to the home turf of K+
channels: neurons, cardiac myocytes, lymphocytes, pancreatic beta cells, and the myriad of cultured mammalian systems where its application will allow for a more nuanced approach to ion channel experimentation in a physiological setting.
Two potential caveats are worth noting when using nonsense suppression to incorporate unnatural amino acids into ion channels. First, both methods generate truncated proteins because, for the time being, the stop codon is essential to guide the charged tRNA to the site of incorporation. For K+
channel expression, these protein fragments are particularly deleterious since they can co-assemble with full-length mutant monomers, resulting in heterotetrameric proteins that are either non-functional or localized to intracellular compartments. In contrast, voltage-gated sodium channel expression is relatively unaffected by truncated proteins since the entire channel protein is formed from a single polypeptide (60
). Second, in a cellular environment, ribosomal compatibility of the unnatural amino acid is a limiting factor when considering unconventional side-chains. In the in vitro
realm, however, advances in protein ligation chemistry have made possible the large scale synthesis of polypeptide chains that contain amino acid variants that would be otherwise difficult, if not impossible, to derive in a cellular setting.
Biochemical approaches to unnatural amino acid incorporation allow for the ligation of two proteins, one of which can be synthetic thus expanding the chemical possibilities of the amino acids used while simultaneously avoiding the stringent control of the ribosome (). A variant of the technique, expressed protein ligation or EPL, takes advantage of a family of novel family of proteins that catalytically ligate recombinant and/or synthetic polypeptides to produce semi-synthetic polypeptides (67
). The general approach, reviewed recently in (68
), has been applied successfully to a number of soluble proteins, yet it’s application to an ion channel is complicated by the substantial experimental hurdles presented by the post-ligation solublization and folding of these integral membrane proteins. These twin challenges not withstanding, the mechanisms of selectivity and gating in the pore region have be investigated with semisynthetic KcsA K+
). Motivation for the use of EPL in the present example can be found upon close inspection of KcsA pore region structures that show the ‘signature’ TVGYG residues alternating between left-handed and right-handed α-helical angles, as if constructed from D and L amino acids, with the two Gly residues adopting dihedral angles typically reserved for D-amino acid counterparts. To test the possibility that Gly-77 truly mimics a D-amino acid, Valiyaveetil and co-workers employed EPL to introduce D-alanine into the KcsA selectivity filter at this site and show that these channels are not only capable of selecting for potassium over lithium, but can bind TEA and charybdotoxin with wild-type affinity, all indicators that the D-alanine is completely tolerated (73
). An additional benefit of the semi-synthetic approach is the potential for the biochemical production of significant quantities of purified unnatural amino acid containing protein, a side benefit that lends itself to structural studies. The ensuing high-resolution crystal structure of D-Ala-77 KcsA was captured in conditions that would normally promote the collapse of the selectivity filter, in the absence of potassium for instance, yet the D-Ala containing channels show a seemingly normal and conducting pore region (70
). These experiments suggest that the endogenous Gly-77 is capable of rotation when the pore region collapses but the methyl side-chains of D-Ala-77 prevent this motion due to the four-fold steric clash that occurs between the other D-Ala methyl groups from other subunits. In sum total, these results explain why this site is unwelcoming to traditional amino acid replacement: glycine is the only naturally occurring residue capable of morphing into the contorted requirements of D-amino acids. The common characterization of the selectivity filter as a protein domain that ‘catalyzes’ the transmembrane flow of potassium rings true when considering that similarly contorted amino acids can also be found in the active sites of enzymes where they sample similar forbidden dihedral space (74
). It seems that K+
channels, like those who study them, will use any tool to get the job done.