Computational elucidation,mutational and hot spot-based designing of potential inhibitors against human acid-sensing ion channels (hASIC-1a) to treat various physiological conditions

Acid-sensing ion channels are ligand/proton-gated ion channels belonging to the family of the degenerin/epithelial Na⁺ channel (DEG/ENaC). They function as a sodium-selective pore for Ca²⁺ entry into neuronal cells during pathological conditions. The blocking of this channel has therapeutic importance, because at basal physiological pH (7.2), it is in a closed state and under a more acidic condition, and the ASIC1a ion channel is activated. To investigate the different states of the hASIC1a channel based on mutational analysis, structure-based virtual screening and molecular dynamics simulation studies. The system showed stability after 30 ns (after 1500 frame), and it was stabilized to an average value around 2.2Å. During the simulation, the ion channel residues in persistent contact with toxin PcTx1 were D237, E238, D347, D351, E219 and E355. These residues are important physiologically for the activation of the channel. From in silico alanine scanning, the significant hotspots obtained in hASIC1 are E344, P347, F352, D351, E355 and E219. From the sitemap analysis, it was evident that the sitemap found one of the active sites at the PcTx1 binding site with a site score of 1.086 and a D-score of 1.035 for hASIC1. We obtained a few promising hits and final potential hits from the virtual screening in hASIC1 that made interactions with the residues in the acidic pocket (E344, P347, F352, D351, E355 and E219). Based on these studies, the hits and scaffolds of potential therapeutic interest against various pathological conditions are associated with hASIC1a for future studies.     

Potentiation of acid-sensing ion channels by sulfhydryl compounds

The acid-sensing ion channels (ASICs) are voltage-independent ion channels activated by acidic extracellular pH. ASICs play a role in sensory transduction, behavior, and acidotoxic neuronal death, which occurs during stroke and ischemia. During these conditions, the extracellular concentration of sulfhydryl reducing agents increases. We used perforated patch-clamp technique to analyze the impact of sulfhydryls on H⁺-gated currents from Chinese hamster ovary (CHO) cells expressing human ASIC1a (hASIC1a). We found that hASIC1a currents activated by pH 6.5 were increased almost twofold by the sulfhydryl-containing reducing agents dithiothreitol (DTT) and glutathione. DTT shifted the pH-dose response of hASIC1a toward a more neutral pH (pH_0.5 from 6.54 to 6.69) and slowed channel desensitization. The effect of reducing agents on native mouse hippocampal neurons and transfected mouse ASIC1a was similar. We found that the effect of DTT on hASIC1a was mimicked by the metal chelator TPEN, and mutant hASIC1a channels with reduced TPEN potentiation showed reduced DTT potentiation. Furthermore, the addition of DTT in the presence of TPEN did not result in further increases in current amplitude. These results suggest that the effect of DTT on hASIC1a is due to relief of tonic inhibition by transition metal ions. We found that all ASICs examined remained potentiated following the removal of DTT. This effect was reversed by the oxidizing agent DTNB in hASIC1a, supporting the hypothesis that DTT also impacts ASICs via a redox-sensitive site. Thus sulfhydryl compounds potentiate H⁺-gated currents via two mechanisms, metal chelation and redox modulation of target amino acids. glutathione; DTT; redox; zinc     

Pyrrolysyl-tRNA synthetase variants reveal ancestral aminoacylation function

Pyrrolysyl-tRNA synthetase (PylRS) is a class IIc aminoacyl-tRNA synthetase that is related to phenylalanyl-tRNA synthetase (PheRS). Genetic selection provided PylRS variants with a broad range of specificity for diverse non-canonical amino acids (ncAAs). One variant is a specific phenylalanine-incorporating enzyme. Structural models of the PylRSamino acid complex show that the small pocket size and π-interaction play an important role in specific recognition of Phe and the engineered PylRS active site resembles that of Escherichia coli PheRS.     

Creation,Optimization, and Use of Semi-Synthetic Organisms that Store and Retrieve Increased Genetic Information

With few exceptions, natural proteins are built from only 20 canonical (proteogenic) amino acids which limits the functionality and accordingly the properties they can possess. Genetic code expansion, i.e. the creation of codons and the machinery needed to assign them to non-canonical amino acids (ncAAs), promises to enable the discovery of proteins with novel properties that are otherwise difficult or impossible to obtain. One approach to expanding the genetic code is to expand the genetic alphabet via the development of unnatural nucleotides that pair to form an unnatural base pair (UBP). Semi-synthetic organisms (SSOs), i.e. organisms that stably maintain the UBP, transcribe its component nucleotides into RNA, and use it to translate proteins, would have available to them new codons and the anticodons needed to assign them to ncAAs. This review summarizes the development of a family of UBPs, their use to create SSOs, and the optimization and application of the SSOs to produce candidate therapeutic proteins with improved properties that are now undergoing evaluation in clinical trials.     

Molecular Cloning and Regional Distribution of a Human Proton Receptor Subunit with Biphasic Functional Properties

Abstract : Small changes of extracellular pH activate depolarizing inward currents in most nociceptive neurons. It has been recently proposed that acid sensitivity of sensory as well as central neurons is mediated by a family of proton-gated cation channels structurally related to Caenorhabditis elegans degenerins and mammalian epithelial sodium channels. We describe here the molecular cloning of a novel human proton receptor, hASIC3, a 531-amino acid-long subunit homologous to rat DRASIC. Expression of homomeric hASIC3 channels in Xenopus oocytes generated biphasic inward currents elicited at pH <5, providing the first functional evidence of a human proton-gated ion channel. Contrary to the DRASIC current phenotype, the fast desensitizing early component and the slow sustained late component differed both by their cationic selectivity and by their response to the antagonist amiloride, but not by their pH sensitivity (pH₅₀ = 3.66 vs. 3.82). Using RT-PCR and mRNA blot hybridization, we detected hASIC3 mRNA in sensory ganglia, brain, and many internal tissues including lung and testis, so hASIC3 gene expression was not restricted to peripheral sensory neurons. These functional and anatomical data strongly suggest that hASIC3 plays a major role in persistent proton-induced currents occurring in physiological and pathological conditions of pH changes, likely through a tissue-specific heteropolymerization with other members of the proton-gated channel family.     

Some theoretical aspects of reprogramming the standard genetic code

Reprogramming of the standard genetic code to include non-canonical amino acids (ncAAs) opens new prospects for medicine, industry, and biotechnology. There are several methods of code engineering, which allow us for storing new genetic information in DNA sequences and producing proteins with new properties. Here, we provided a theoretical background for the optimal genetic code expansion, which may find application in the experimental design of the genetic code. We assumed that the expanded genetic code includes both canonical and non-canonical information stored in 64 classical codons. What is more, the new coding system is robust to point mutations and minimizes the possibility of reversion from the new to old information. In order to find such codes, we applied graph theory to analyze the properties of optimal codon sets. We presented the formal procedure in finding the optimal codes with various number of vacant codons that could be assigned to new amino acids. Finally, we discussed the optimal number of the newly incorporated ncAAs and also the optimal size of codon groups that can be assigned to ncAAs.     

An arginine residue in the outer segment of hASIC1a TM1 affects both proton affinity and channel desensitization

Acid-sensing ion channels (ASICs) respond to changes in pH in the central and peripheral nervous systems and participate in synaptic plasticity and pain perception. Understanding the proton-mediated gating mechanism remains elusive despite the of their structures in various conformational states. We report here that R64, an arginine located in the outer segment of the first transmembrane domain of all three isoforms of mammalian ASICs, markedly impacts the apparent proton affinity of activation and the degree of desensitization from the open and preopen states. Rosetta calculations of free energy changes predict that substitutions of R64 in hASIC1a by aromatic residues destabilize the closed conformation while stabilizing the open conformation. Accordingly, F64 enhances the efficacy of proton-mediated gating of hASIC1a, which increases the apparent pH₅₀ and facilitates channel opening when only one or two subunits are activated. F64 also lengthens the duration of opening events, thus keeping channels open for extended periods of time and diminishing low pH-induced desensitization. Our results indicate that activation of a proton sensor(s) with pH₅₀ equal to or greater than pH 7.2–7.1 opens F64hASIC1a, whereas it induces steady-state desensitization in wildtype channels due to the high energy of activation imposed by R64, which prevents opening of the pore. Together, these findings suggest that activation of a high-affinity proton-sensor(s) and a common gating mechanism may mediate the processes of activation and steady-state desensitization of hASIC1a.     

On the putative binding site of RFamide-family neuropeptides from the western Atlantic clam Sunray Venus and cephalopods on acid-sensing ion channels. An automated docking and molecular-dynamics study with hASIC1a homology model

Investigated here are interactions of C-terminal amidated peptides with the hASIC1a acid-sensing ion channel. The peptides comprise endogenous FMRFa, present in the western Atlantic clam Sunray Venus, and FIRFa, present in cephalopods, as well as non-endogenous ones for comparison. The interaction is investigated by automated docking. The resulting key hASIC1a-FMRFa complex, set in a lipidic POPC (=1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) membrane surrounded by H(2) O and Na(+) -neutralized, was also investigated by molecular dynamics. It was observed that all investigated peptides become encapsulated into the ion channel, on one side by the thumb and finger of a subunit, and, on the opposite side, by the knuckle and β-ball of a second subunit. The third subunit is not involved. This is much the same binding site that was disclosed previously by both a similar computational approach, and electrophysiological and binding experiments for the hASIC1a ion channel-blocker tarantula toxin PCTX1. This paves the way to a better understanding of the role of these peptides in invertebrates.     

Two splice variants derived from a Drosophila melanogaster candidate ClC gene generate ClC-2-type Cl- channels

Members of the ClC family of membrane proteins have been found in a variety of species and they can function as Cl- channels or Cl-/H+ antiporters. Three potential ClC genes are present in the Drosophila melanogaster genome. Only one of them shows homology with a branch of the mammalian ClC genes that encode plasma membrane Cl- channels. The remaining two are close to mammalian homologues coding for intracellular ClC proteins. Using RT-PCR we have identified two splice variants showing highest homology (41% residue identity) to the mammalian ClC-2 chloride channel. One splice variant (DmClC-2S) is expressed in the fly head and body and an additional, larger variant (DmClC-2L) is only present in the head. Both putative Drosophila channels conserve key features of the ClC channels cloned so far, including residues conforming the selectivity filter and C-terminus CBS domains. The splice variants differ in a stretch of 127 aa at the intracellular C-terminal portion separating cystathionate beta synthase (CBS) domains. Expression of either Drosophila ClC-2 variant in HEK-293 cells generated inwardly rectifying Cl- currents with similar activation and deactivation characteristics. There was great similarity in functional characteristics between DmClC-2 variants and their mammalian counterpart, save for slower opening kinetics and faster closing rate. As CBS domains are believed to be sites of regulation of channel gating and trafficking, it is suggested that the extra amino acids present between CBS domains in DmClC-2L might endow the channel with a differential response to signals present in the fly cells where it is expressed.     

Rationally evolving tRNAPyl for efficient incorporation of noncanonical amino acids

Genetic encoding of noncanonical amino acids (ncAAs) into proteins is a powerful approach to study protein functions. Pyrrolysyl-tRNA synthetase (PylRS), a polyspecific aminoacyl-tRNA synthetase in wide use, has facilitated incorporation of a large number of different ncAAs into proteins to date. To make this process more efficient, we rationally evolved tRNA^Pyl to create tRNA^Pyl-opt with six nucleotide changes. This improved tRNA was tested as substrate for wild-type PylRS as well as three characterized PylRS variants (N^ϵ-acetyllysyl-tRNA synthetase [AcKRS], 3-iodo-phenylalanyl-tRNA synthetase [IFRS], a broad specific PylRS variant [PylRS-AA]) to incorporate ncAAs at UAG codons in super-folder green fluorescence protein (sfGFP). tRNA^Pyl-opt facilitated a 5-fold increase in AcK incorporation into two positions of sfGFP simultaneously. In addition, AcK incorporation into two target proteins (Escherichia coli malate dehydrogenase and human histone H3) caused homogenous acetylation at multiple lysine residues in high yield. Using tRNA^Pyl-opt with PylRS and various PylRS variants facilitated efficient incorporation of six other ncAAs into sfGFP. Kinetic analyses revealed that the mutations in tRNA^Pyl-opt had no significant effect on the catalytic efficiency and substrate binding of PylRS enzymes. Thus tRNA^Pyl-opt should be an excellent replacement of wild-type tRNA^Pyl for future ncAA incorporation by PylRS enzymes.     

Functional properties and toxin pharmacology of a dorsal root ganglion sodium channel viewed through its voltage sensors

The voltage-activated sodium (Nav) channel Nav1.9 is expressed in dorsal root ganglion (DRG) neurons where it is believed to play an important role in nociception. Progress in revealing the functional properties and pharmacological sensitivities of this non-canonical Nav channel has been slow because attempts to express this channel in a heterologous expression system have been unsuccessful. Here, we use a protein engineering approach to dissect the contributions of the four Nav1.9 voltage sensors to channel function and pharmacology. We define individual S3b-S4 paddle motifs within each voltage sensor, and show that they can sense changes in membrane voltage and drive voltage sensor activation when transplanted into voltage-activated potassium channels. We also find that the paddle motifs in Nav1.9 are targeted by animal toxins, and that these toxins alter Nav1.9-mediated currents in DRG neurons. Our results demonstrate that slowly activating and inactivating Nav1.9 channels have functional and pharmacological properties in common with canonical Nav channels, but also show distinctive pharmacological sensitivities that can potentially be exploited for developing novel treatments for pain.     

Performance analysis of orthogonal pairs designed for an expanded eukaryotic genetic code

Background

The suppression of amber stop codons with non-canonical amino acids (ncAAs) is used for the site-specific introduction of many unusual functions into proteins. Specific orthogonal aminoacyl-tRNA synthetase (o-aaRS)/amber suppressor tRNA_CUA pairs (o-pairs) for the incorporation of ncAAs in S. cerevisiae were previously selected from an E. coli tyrosyl-tRNA synthetase/tRNA_CUA mutant library. Incorporation fidelity relies on the specificity of the o-aaRSs for their ncAAs and the ability to effectively discriminate against their natural substrate Tyr or any other canonical amino acid.

Methodology/Principal Findings

We used o-pairs previously developed for ncAAs carrying reactive alkyne-, azido-, or photocrosslinker side chains to suppress an amber mutant of human superoxide dismutase 1 in S. cerevisiae. We found worse incorporation efficiencies of the alkyne- and the photocrosslinker ncAAs than reported earlier. In our hands, amber suppression with the ncAA containing the azido group did not occur at all. In addition to the incorporation experiments in S. cerevisiae, we analyzed the catalytic properties of the o-aaRSs in vitro. Surprisingly, all o-aaRSs showed much higher preference for their natural substrate Tyr than for any of the tested ncAAs. While it is unclear why efficiently recognized Tyr is not inserted at amber codons, we speculate that metabolically inert ncAAs accumulate in the cell, and for this reason they are incorporated despite being weak substrates for the o-aaRSs.

Conclusions/Significance

O-pairs have been developed for a whole plethora of ncAAs. However, a systematic and detailed analysis of their catalytic properties is still missing. Our study provides a comprehensive scrutiny of o-pairs developed for the site-specific incorporation of reactive ncAAs in S. cerevisiae. It suggests that future development of o-pairs as efficient biotechnological tools will greatly benefit from sound characterization in vivo and in vitro in parallel to monitoring intracellular ncAA levels.     

Variants of TRP ion channel mRNA present in horseshoe crab ventral eye and brain

Transient receptor potential (TRP) channels mediate light-induced Ca(2+) entry and the electrical response in Drosophila photoreceptors. The role of TRP channels in other invertebrate photoreceptors is unknown, particularly those, exemplified by Limulus ventral eye photoreceptors, in which calcium release from intracellular stores is prominent. We have amplified cDNA encoding three variants of a Limulus TRP channel. LptrpA and LptrpBencode proteins of 896 and 923 amino acids, differing by a 27 amino acid insert within the C-terminus. LptrpC encodes an alternative 63 amino acid sequence in the pore domain compared with LptrpB. LptrpB and LptrpC are present in ventral eye mRNA, while LptrpA is only present in brain mRNA. In situ hybridization indicates the presence of Lptrp in photoreceptors of the Limulus ventral eye. Some canonical TRP channels can be activated by diacylglycerol analogs. Injection of a diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG), into Limulus photoreceptors can activate an inward current with electrical characteristics similar to the light-activated current. However, simultaneous elevation of cytosolic calcium concentration appears to be necessary. Illumination attenuates the response to OAG injections and vice versa. These results provide molecular and pharmacological evidence for a TRP channel in Limulus ventral eye that may contribute to the light-sensitive conductance.     

A flexible GAS belt responds to pore mutations changing the ion selectivity of proton-gated channels

Proton-gated ion channels conduct mainly Na⁺ to induce postsynaptic membrane depolarization. Finding the determinants of ion selectivity requires knowledge of the pore structure in the open conformation, but such information is not yet available. Here, the open conformation of the hASIC1a channel was computationally modeled, and functional effects of pore mutations were analyzed in light of the predicted structures. The open pore structure shows two constrictions of similar diameter formed by the backbone of the GAS belt and, right beneath it, by the side chains of H28 from the reentrant loop. Models of nonselective mutant channels, but not those that maintain ion selectivity, predict enlargement of the GAS belt, suggesting that this motif is quite flexible and that the loss of stabilizing interactions in the central pore leads to changes in size/shape of the belt. Our results are consistent with the “close-fit” mechanism governing selectivity of hASIC1a, wherein the backbone of the GAS substitutes at least part of the hydration shell of a permeant ion to enable crossing the pore constriction.     

Biosynthesis and biotechnological application of non-canonical amino acids: Complex and unclear

Compared with the better-studied canonical amino acids, the distribution, metabolism and functions of natural non-canonical amino acids remain relatively obscure. Natural non-canonical amino acids have been mainly discovered in plants as secondary metabolites that perform diversified physiological functions. Due to their specific characteristics, a broader range of natural and artificial non-canonical amino acids have recently been applied in the development of functional materials and pharmaceutical products. With the rapid development of advanced methods in biotechnology, non-canonical amino acids can be incorporated into peptides, proteins and enzymes to improve the function and performance relative to their natural counterparts. Therefore, biotechnological application of non-canonical amino acids in artificial bio-macromolecules follows the central goal of synthetic biology to: create novel life forms and functions. However, many of the non-canonical amino acids are synthesized via chemo- or semi-synthetic methods, and few non-canonical amino acids can be synthesized using natural in vivo pathways. Therefore, further research is needed to clarify the metabolic pathways and key enzymes of the non-canonical amino acids. This will lead to the discovery of more candidate non-canonical amino acids, especially for those that are derived from microorganisms and are naturally bio-compatible with chassis strains for in vivo biosynthesis. In this review, we summarize representative natural and artificial non-canonical amino acids, their known information regarding associated metabolic pathways, their characteristics and their practical applications. Moreover, this review summarizes current barriers in developing in vivo pathways for the synthesis of non-canonical amino acids, as well as other considerations, future trends and potential applications of non-canonical amino acids in advanced biotechnology.     

Structure-based non-canonical amino acid design to covalently crosslink an antibody–antigen complex

Engineering antibodies to utilize non-canonical amino acids (NCAA) should greatly expand the utility of an already important biological reagent. In particular, introducing crosslinking reagents into antibody complementarity determining regions (CDRs) should provide a means to covalently crosslink residues at the antibody–antigen interface. Unfortunately, finding the optimum position for crosslinking two proteins is often a matter of iterative guessing, even when the interface is known in atomic detail. Computer-aided antibody design can potentially greatly restrict the number of variants that must be explored in order to identify successful crosslinking sites. We have therefore used Rosetta to guide the introduction of an oxidizable crosslinking NCAA, l-3,4-dihydroxyphenylalanine (l-DOPA), into the CDRs of the anti-protective antigen scFv antibody M18, and have measured crosslinking to its cognate antigen, domain 4 of the anthrax protective antigen. Computed crosslinking distance, solvent accessibility, and interface energetics were three factors considered that could impact the efficiency of l-DOPA-mediated crosslinking. In the end, 10 variants were synthesized, and crosslinking efficiencies were generally 10% or higher, with the best variant crosslinking to 52% of the available antigen. The results suggest that computational analysis can be used in a pipeline for engineering crosslinking antibodies. The rules learned from l-DOPA crosslinking of antibodies may also be generalizable to the formation of other crosslinked interfaces and complexes.     

Using a quadruplet codon to expand the genetic code of an animal

Genetic code expansion in multicellular organisms is currently limited to the use of repurposed amber stop codons. Here, we introduce a system for the use of quadruplet codons to direct incorporation of non-canonical amino acids in vivo in an animal, the nematode worm Caenorhabditis elegans. We develop hybrid pyrrolysyl tRNA variants to incorporate non-canonical amino acids in response to the quadruplet codon UAGA. We demonstrate the efficiency of the quadruplet decoding system by incorporating photocaged amino acids into two proteins widely used as genetic tools. We use photocaged lysine to express photocaged Cre recombinase for the optical control of gene expression and photocaged cysteine to express photo-activatable caspase for light inducible cell ablation. Our approach will facilitate the routine adoption of quadruplet decoding for genetic code expansion in eukaryotic cells and multicellular organisms.     

Different structural requirements for functional ion pore transplantation suggest different gating mechanisms of NMDA and kainate receptors

Although considerable progress has been made in characterizing the physiological function of the high-affinity kainate (KA) receptor subunits KA1 and KA2, no homomeric ion channel function has been shown. An ion channel transplantation approach was employed in this study to directly test if homomerically expressed KA1 and KA2 pore domains are capable of conducting currents. Transplantation of the ion pore of KA1 or KA2 into GluR6 generated perfectly functional ion channels that allowed characterization of those electrophysiological and pharmacological properties that are determined exclusively by the ion pore of KA1 or KA2. This demonstrates for the first time that KA1 and KA2 ion pore domains are intrinsically capable of conducting ions even in homomeric pore assemblies. NMDA receptors, similar to KA1- or KA2-containing receptors, function only as heteromeric complexes. They are composed of NR1 and NR2 subunits, which both are non-functional when expressed homomerically. In contrast to NR1, the homomeric NR2B ion pore failed to translate ligand binding into pore opening when transplanted into GluR6. Similarly, heteromeric coexpression of the ion channel domains of both NR1 and NR2 inserted into GluR6 failed to produce functional channels. Therefore, we conclude that the mechanism underlying the ion channel opening in the obligatorily heterotetrameric NMDA receptors differs significantly from that in the facultatively heterotetrameric alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate and KA receptors.     

Stable and functional expression of the calcium channel alpha 1 subunit from smooth muscle in somatic cell lines.

Voltage-activated calcium channels are membrane spanning proteins that allow the controlled entry of Ca2+ into the cytoplasm of cells. The principal channel forming subunit of an L-type calcium channel is the alpha 1 subunit. Transfection of Chinese hamster ovary (CHO) cells with complementary DNA encoding the calcium channel alpha 1 subunit from smooth muscle led to the expression of functional calcium channels which bind calcium channel blockers and show the voltage-dependent activation and slow inactivation and unitary current conductance characteristic of calcium channels in smooth muscle. The currents mediated by these channels are sensitive towards dihydropyridine-type blockers and agonists indicating that the calcium channel blocker receptor sites were present in functional form. The smooth muscle alpha 1 subunit cDNA alone is sufficient for stable expression of functional calcium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.