Genomic expansions of simple tandem repeats can give rise to toxic RNAs that contain expanded repeats. In myotonic dystrophy, the expression of expanded CUG repeats (CUGexp) causes abnormal regulation of alternative splicing and neuromuscular dysfunction. We used a transgenic mouse model to show that derangements of myotonic dystrophy are reversed by a morpholino antisense oligonucleotide, CAG25, that binds to CUGexp RNA and blocks its interaction with muscleblind-like 1 (MBNL1), a CUGexp-binding protein. CAG25 disperses nuclear foci of CUGexp RNA and reduces the overall burden of this toxic RNA. As MBNL1 is released from sequestration, the defect of alternative splicing regulation is corrected, thereby restoring ion channel function. These findings suggest an alternative use of antisense methods, to inhibit deleterious interactions of proteins with pathogenic RNAs.
Abstract Acid‐sensing ion channels (ASICs) are important players in detecting extracellular acidification throughout the brain and body. ASICs have large extracellular domains containing two regions replete with acidic residues: the acidic pocket, and the palm domain. In the resting state, the acidic pocket is in an expanded conformation but collapses in low pH conditions as the acidic side chains are neutralized. Thus, extracellular acidification has been hypothesized to collapse the acidic pocket that, in turn, ultimately drives channel activation. However, several observations run counter to this idea. To explore how collapse or mobility of the acidic pocket is linked to channel gating, we employed two distinct tools. First, we incorporated the photocrosslinkable noncanonical amino acids (ncAAs) 4‐azido‐L‐phenylalanine (AzF) or 4‐benzoyl‐L‐phenylalanine (BzF) into several positions in the acidic pocket. At both E315 and Y318, AzF incorporation followed by UV irradiation led to right shifts in pH response curves and accelerations of desensitization and deactivation, consistent with restrictions of acidic pocket mobility destabilizing the open state. Second, we reasoned that because Cl − ions are found in the open and desensitized structures but absent in the resting state structures, Cl − substitution would provide insight into how stability of the pocket is linked to gating. Anion substitution resulted in faster deactivation and desensitization, consistent with the acidic pocket regulating the stability of the open state. Taken together, our data support a model where acidic pocket collapse is not essential for channel activation. Rather, collapse of the acidic pocket influences the stability of the open state of the pore.
Myotonic dystrophy type 1 (DM1) involves misregulated alternative splicing for specific genes. We used exon or nucleotide deletion to mimic altered splicing of genes central to muscle excitation-contraction coupling in mice. Mice with forced skipping of exon 29 in the CaV1.1 calcium channel combined with loss of ClC-1 chloride channel function displayed markedly reduced lifespan, whereas other combinations of splicing mimics did not affect survival. The Ca2+/Cl– bi-channelopathy mice exhibited myotonia, weakness, and impairment of mobility and respiration. Chronic administration of the calcium channel blocker verapamil rescued survival and improved force generation, myotonia, and respiratory function. These results suggest that Ca2+/Cl– bi-channelopathy contributes to muscle impairment in DM1 and is potentially mitigated by common clinically available calcium channel blockers.
The CDKL5 deficiency disorder (CDD) is a severe neurodevelopmental disorder characterized by early-onset epilepsy, intellectual disability, motor and visual dysfunctions. The causative gene is CDKL5, which codes for a kinase required for brain development. There is no cure for CDD patients; treatments are symptomatic and focus mainly on seizure control. Several pathogenic variants are loss-of-function, but recent studies suggest that the CDD phenotype is sensitive to the CDKL5 gene dosage. Therefore, mRNA-targeted correction strategies that respect the physiological regulation of CDKL5 could be a valid alternative to augmentative gene therapy. Nonsense mutations cause ~ 11% of CDD cases, and these patients might benefit from readthrough therapies. We proved that drug-mediated readthrough efficiently suppresses premature CDKL5 nonsense codons, but the recoded kinase remained highly hypomorphic, curtailing the translational value of this pharmacological approach. In this study we explored if the recently developed Anticodon-edited tRNAs (ACE-tRNAs) offer an alternative readthrough strategy for CDD. Transfecting cells expressing different CDKL5 nonsense variants, we demonstrated that ACE-tRNAs efficiently restore full-length kinase synthesis. The recoded CDKL5 is correctly localized and catalytically active, thereby bringing tRNA-based therapy back into the spotlight for future investigations to assess the efficacy of this approach in correcting the pathological phenotype of CDD.
Full text Figures and data Side by side Abstract Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract 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. https://doi.org/10.7554/eLife.18976.001 Introduction C-type inactivation, also termed ‘slow-inactivation’ due to its delayed kinetics, in voltage-gated potassium channels describes a phenomenon by which ionic conductance through the channel pore is decreased in the presence of continued depolarizing stimulus (Choi et al., 1991; Hoshi et al., 1991). In a physiological setting, this mechanism represents a form of activity-dependent macromolecular memory that can alter action-potential firing rates (Aldrich et al., 1979; Roeper et al., 1997). The basis for C-type inactivation has been investigated with functional, structural and computational approaches, which have produced a consensus mechanism whereby channels become non-conducting due to local perturbations in the selectivity filter (Yellen et al., 1994; Baukrowitz and Yellen, 1995; Kiss and Korn, 1998; Perozo et al., 1998; Roux and MacKinnon, 1999; Bernèche and Roux, 2001; Cordero-Morales et al., 2007; Panyi and Deutsch, 2007; Cuello et al., 2010; Ostmeyer et al., 2013; Thomson et al., 2014). However, the nature of the conformational change and role of specific residues remain an area of active experimentation and debate (Devaraneni et al., 2013; Hoshi and Armstrong, 2013). We previously identified an intra- and inter-subunit hydrogen bond network that controls the rate of entry into the non-conducting pore conformation (Pless et al., 2013). Our study employed nonsense suppression in the Xenopus laevis oocyte expression system to genetically encode a variety of synthetic noncanonical amino acids (ncAAs). In the context of the Xenopus laevis oocyte, the cRNA of the target protein contains an introduced stop codon (often amber, TAG) that is subsequently suppressed by a co-injected orthogonal tRNA that has been misacylated in vitro with the ncAA. This approach was used to encode a tryptophan homologue, 2-amino-3-indol-1-yl-propionic acid (referred to as Ind) that fails to participate in hydrogen bonding at position 434, a site previously identified as being important for channel inactivation. Our original efforts failed to determine the functional role of individual hydrogen bonds with Ind because subunit concatenation in the tetrameric construct resulted in a significant loss of expressing channels, an outcome that prevented the application of nonsense suppression (Pless et al., 2013). Reduced channel expression is a common challenge of the nonsense suppression method where amino acid incorporation varies greatly from position to position within a single reading frame and between channel families. Furthermore, given the central role of the Trp434-Asp447 hydrogen bond in the mechanism of C-type inactivation, it was not possible to simultaneously encode Ind within each of the four subunits of the channel because this resulted in a non-conducting phenotype which lacked the relatively high expression levels needed to measure gating currents which originate from the movement of the channel voltage-sensor in the transmembrane electric field (Perozo et al., 1993). Thus, we concluded that while one can control the stoichiometry of individual subunit composition in a concatenated construct, their low expression impeded the use of nonsense suppression in combination with this approach. In our original publication, this shortcoming was partially overcome by combining WT cRNA with Ind-tRNA and Trp434TAG cRNA within an individual oocyte. This experimental approach resulted in predicted accelerated, but complex, inactivation kinetics consistent with a mixed population of channels with 1, 2 or 3 Ind434-containing monomers (see Figure 2F, Pless et al., 2013, eLife). In the present study, we examined the mechanism of Ind-induced inactivation by generating a new approach to control subunit stoichiometry to test individual hydrogen bond contributions in Shaker channel C-type inactivation and more generally, for the encoding of multiple ncAA within a single channel or an ion channel complex in the application of nonsense suppression. Results Molecular dynamic simulations of Ind and the Trp434-Asp447 hydrogen bond Targeted replacement of Trp434 with the isosteric ncAA Ind was used previously to highlight the general importance of the indole hydrogen bond of this highly conserved Trp residue. The Ind amino acid has seen limited use in structure-function studies for hydrogen bond testing (Lacroix et al., 2012; Pless et al., 2013, 2014; Kim et al., 2015; Zhang et al., 2015) and none thus far in structural or computational studies that would inform on its tolerance once encoded within a protein. For instance, it is not known if the Ind substitution would specifically limit hydrogen bonding at the indole nitrogen atom (the purpose for which it was designed) or could it also produce a new side-chain orientation that itself alters protein function. A computational approach based on molecular dynamics (MD) simulations was therefore employed to examine the local dynamics of the Ind side-chain and its relation to the intra-subunit hydrogen bond accepting side-chain Asp447. These MD simulations reveal key conformational and dynamic differences between wild type (WT) and Trp434Ind of the Shaker channel selectivity filter in the conductive state (Long et al., 2005) (Figure 1A). In WT Shaker, a hydrogen bond is formed between Trp434 and Asp447, restraining the two residues close to each other during a large fraction of a 500 ns trajectory (Figure 1C). In the absence of the Asp447-Trp434 hydrogen bond in the Trp434Ind Shaker, there is an increased distance between these two residues in all of the four subunits (Figure 1C), which is associated with a flipping of the Asp447 side-chain toward the extracellular bulk solution (Figure 1B). See also Supplemental Videos 1 and 2 for wildtype W434 and W434Ind channels which depict 150 ns vignettes of the full molecular dynamics simulations. Figure 1 Download asset Open asset Conformational dynamics of wild type and Trp434Ind Shaker channel. (A) Position of the Trp434 –Asp447 H-bond. (B) Time series of dihedral angles χ1 (N–Cα–Cβ–Cγ) of Arp447 for wild type (left) and Trp434Ind (right) demonstrating the flipping of the Arp447 side-chains during the simulations. (C) Time series of distance between Asp447 and Trp434 in four subunits characterizing the local conformational dynamics during equilibrium simulations for wild type (left) and Trp434Ind (right) of Shaker. The distance is measured between Asp447 (Cγ) and Trp434 (Nε1) or equivalent atom in the same position in Trp434Ind. (D) Top panels, typical conformations for protein and water molecules and occupancy (20%) map of water molecules around selectivity filter of subunit C. Bottom panels, 2D average occupancy map for all four subunits during 500ns MD simulations. The x-axis describes the radius to the center of the selectivity filter, and the y-axis is the z-coordinate of water molecules. In all simulations the selectivity filter in the conductive state. Videos 1 and 2 indicate 150 ns vignettes of the simulation for the W434-D447 and W434Ind-D447 in the presence shown water molecules. In each case, a subunit of Shaker (residues 433 to 447) is shown in new-cartoon representation, and side chains of W434 and D447 are shown in sticks, and water molecules are in VDW representations. https://doi.org/10.7554/eLife.18976.002 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg In the WT a hydrogen bond is formed between Trp434 and Asp447 during a large fraction of MD simulation, which blocks the exchange of water molecules between extracellular bulk and the water binding pocket behind selectivity filter. Upon rare occasion the D447 H-bond is broken spontaneously but quickly reforms. https://doi.org/10.7554/eLife.18976.003 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg In the Trp434Ind Shaker, there is a flipping of the Asp447 side-chain toward the extracellular bulk solution, promoting the formation of a continuous connection between the bulk solution and the water molecules in the buried pocket. https://doi.org/10.7554/eLife.18976.004 The breaking of the Asp447-Trp434 hydrogen bond in the Trp434Ind mutant has two major effects in the vicinity of the selectivity filter. First, the formation of a hydrogen bond between Asp447-Trp434 in the wild type blocks the exchange of water molecules between extracellular bulk and the water binding pocket behind the selectivity filter, as shown in the water occupancy map in Figure 1D. In the absence of this hydrogen bond in Trp434Ind, the increased spatial separation of Asp447 and Trp434Ind promotes the formation of a continuous connection between the bulk solution and the water molecules in the buried pocket. The easier accessibility of the buried water-binding pocket probably accelerates C-type inactivation of Shaker. Second, the flipping of Asp447 leads to an additional effect to the selectivity filter. Considering Asp447 in each subunit carries a negative charge, the reorientation of the Asp447 side-chain from facing inside to outside (Figure 1C) perturbs the local electrostatic potential at the outer end of the selectivity filter, altering the binding affinity of potassium ions in multiple binding sites, and is likely to shift the relative stability between conductive and nonconductive states. A strategy for defining the stoichiometry of multimeric protein complexes An engineered protein approach was designed to circumvent issues raised with expressing Shaker monomer concatemers: complementary split inteins (Wu et al., 1998; Muralidharan and Muir, 2006), were incorporated into the N- or C- termini of Shaker monomers (Figure 2A). Split inteins drive a post-translational event involving precise excision of the intein sequence and formation of a normal peptide bond between the flanking sequences (N- and C-exteins) (Saleh and Perler, 2006). We reasoned that Shaker monomers would form split intein-dependent dimers, and Shaker channels would consist of dimers of dimers. In order to exclude nonconforming Shaker monomers from the surface membrane, we encoded endoplasmic reticulum retention/retrieval (ERret) motifs which flank the split inteins (Figure 2A) that would be removed following the intein ligation process (Figure 2B). We used the Kir6.2 ERret motif because it works in a variety of eukaryotic cell types, has strong retention properties, and functions independently from its position within a protein (Zerangue et al., 1999). Oocytes injected with only one of the monomer cRNAs did not show measurable voltage-dependent K+ selective currents (Figure 2A, lower panels). This is presumably due to the retention of the monomers within the ER as a result of the appended ERret motifs. In contrast, co-expression of Shaker monomers with complementary split inteins resulted in robust outward voltage-dependent potassium currents due to the nature of the split intein protein ligation mechanism. The formation of tetrameric dimer-of-dimer complexes with no ERret motif allows trafficking of Shaker channels from the ER to the plasma membrane. The level of expressed potassium current was stable over a large range of injected Shaker monomer cRNA concentrations (Figure 2B) suggesting a limiting cellular component in channel assembly and/or trafficking. This method allows for control of subunit stoichiometry and the possibility for encoding multiple Ind side-chains within a single channel tetramer by placing the introduced TAG mutation within one (or both) of the two complementary fragments that form a dimer. Figure 2 Download asset Open asset Attachment of ER retention/retrieval motifs paired with split inteins results in co-dependent surface membrane expression of Shaker channels. (A) Expression of either Shaker channel with C-terminal appended Kir6.2 ER retention/retrieval motif (ERret; yellow) with N-intein (red) (A, left) or Shaker channel with N-terminal appended Kir6.2 ER retention/retrieval motif (yellow) with C-intein (green) (A, right), resulted in negligible ionic current (A, lower panels).Representative currents in lower panel A, are raw capacitance and ionic currents that are blanked above and below 25 and −5µA, respectively. P/4 subtracted currents are shown in lower panel B to display normal channel kinetics observed following expression of engineered ERret-intein Shaker channels. See Materials and methods for ER-retention/retrieval and intein sequences. (B) Co-expression of Shaker N- and C-terminal ERret-intein channels resulted in surface membrane trafficking of Shaker channels and robust ionic current (lower panel) measured by TEVC. (C) Injection of increased amounts of C-terminal (filled circles) or N-terminal (open circles) ERret-intein-tagged Shaker cRNA resulted in insignificant surface expression of Shaker channels as measured with TEVC. Co-injection of increased amounts of N- and C-terminal ERret-intein cRNA resulted in saturated ionic current (filled triangles). Number of observations where two Shaker monomers are expressed are indicated in parentheses above symbols. Average values where only one Shaker monomer was expressed were composed of 2–10 observations. https://doi.org/10.7554/eLife.18976.005 Using ERret-Intein protein engineering to encode two ncAA within a Shaker channel In order to test cooperativity of the hydrogen bond at position 434, the ERret-intein approach was used to express Shaker channels with two Ind residues at position 434 per channel (Figure 3). In experiments where both WT and Trp434TAG complimentary ERret-intein cRNAs and Trp-tRNA were injected, robust WT-like currents were observed, demonstrating successful Trp incorporation (Figure 3A, left). Conversely, when Ind-tRNA was expressed within the C-terminal Shaker monomer of each dimer, C-type inactivation was significantly accelerated, consistent with the breakage of two of the four hydrogen bonds around the central permeation pathway (Figure 3A, right) while voltage dependence of activation was not altered (Figure 3B, Table 1). At +20 mV, WT Shaker channels had an inactivation time constant of 3900 +/- 0.2 milliseconds (first-order exponential) compared to 529 +/-141 and 142 +/-14 milliseconds when one or two hydrogen bonds were broken through Ind incorporation, respectively (Figure 3C). Figure 3 Download asset Open asset Use of ERret-intein tagged Shaker monomers allow for stoichiometric expression of Ind. (A) Normalized representative currents for Trp434Trp (WT) (left) and Trp434Ind (right). A schematic of the stoichiometry of the incorporated amino acids Trp (white) and Ind (black) is shown below the traces. (B) Average voltage dependence of normalized currents for Trp434Trp (WT) (open circles; n = 5) and Trp434Ind (filled circles, n = 9). (C) Average inactivation rates at +20 mV of WT Shaker, Trp434Phe (first monomer) four monomer concatemer, and Trp434Ind ERret-intein constructs. https://doi.org/10.7554/eLife.18976.006 Table 1 Shaker and Nav1.4 Channel activation parameters. https://doi.org/10.7554/eLife.18976.007 Injected cRNAV ½ (mV)K (mV)Shaker Inteins WT-WT (0.25 ng cRNA)−20.74 ± 2.27 (7) 11.42 ± 0.55 (7) WT-Trp434Trp (0.25 ng cRNA)−23.29 ± 1.76 (9) 9.38 ± 0.85 (9) WT-Trp434Ind (0.25 ng cRNA)−17.84 ± 2.17 (5) 7.75 ± 0.74 (5) Nav1.4 Full-length WT Nav1.4 (2.5 ng cRNA)−25.63 ± 0.71 (5) 2.70 ± 0.24 (5) Split WT Nav1.4 (2.5 ng cRNA)−25.97 ± 1.21 (6) 2.86 ± 0.23 (6) Y401TAG (FH) + WT (BH) + tRNAPhe (25 ng cRNA)−22.78 ± 0.54 (8) 3.99 ± 0.13 (8) WT (FH) + F1304TAG (BH) + tRNA(Phe) (25 ng cRNA)−25.22 ± 1.12 (7) 4.09 ± 0.19 (7) Y401TAG (FH) + F1304TAG (BH) + tRNA(Phe) (50 ng cRNA)−24.22 ± 0.55 (7) 3.78 ± 0.18 (7) In order to examine the general utility of the ERret-intein sequences, they were constructed within two-domain hemi-channels of the skeletal muscle isoform of the voltage-gated sodium channel NaV1.4. Previously, it has been shown that both sodium and calcium channels are amenable to functional complementation via expression of ‘split’ hemi-channels consisting of DI-DIII and DIV or DI-II and DIII-DIV, respectively (Stühmer et al., 1989; Ahern et al., 2001). This result suggests that sodium and calcium channels need not be tethered on a single polypeptide to form a functional tetrameric channel. We therefore reasoned that co-assembly of sodium channel domains I-II and III-IV would likely bring ERret-intein sequences on the C-terminus of DI-II and the N-terminus of DIII-IV in close proximity and promote intein dependent cleavage of the ERret sequences and formation of a peptide bond between domains II and III (Figure 4A). To this end, cRNA for Nav1.4 DI-II and DIII-IV ERret-intein containing hemi-channel fragments were expressed in Xenopus oocyte and subsequent currents were recorded by TEVC. As with the Shaker intein constructs, expression of either individual fragment did not result in expressed ionic currents (Figure 4D; triangles and squares). However, co-expression of the split NaV1.4 halves resulted in robust inward voltage-gated sodium currents (Figure 4C– D, diamonds) similar to WT full-length channels (Figure 4D, circles). Notably, full-length sodium channels produced from the ERret-inteins had biophysical properties that were indistinguishable from WT sodium channels, p>0.01, Table 1. To examine the general versatility of the approach, we expressed the NaV1.4hemi-channel construct cDNA by transient transfection into HEK293 cells and examined the ligation efficiency by Western blot using C-terminal HA epitopes that are on both N- and C-channel fragments (Figure 4B). In the case of the DI-II-HA construct, this epitope is cleaved out during the in-cell ligation reaction but the full-length channel can still be identified by the remaining C-terminal HA epitope on the DIII-IV protein (see methods for protein sequence and construct design). The Western blot data show that each hemi-channel is robustly expressed, producing bands of ~150 kDa (Figure 4B, lane 2) and ~110 kDa (Figure 4B, lane 3) for the DI-II-HA and DIII-DIV-HA truncated constructs, respectively. Co-expression of the channel halves resulted in the formation of a ~250 kDa band (lanes 4 and 5) similar to WT NaV1.4 full-length channels (lane 1). The efficiency of protein ligation was similar to that of intein ligated Ca2+ channel hemi-channel fragments where also, interestingly, the fragment corresponding to the DIII-IV construct was limiting (Subramanyam et al., 2013). To rule out the possibility that the ligation reaction occurred during sample preparation, i.e after the fragments had been solubilized, cell lysates from NaV1.4 DI-II or DIII-IV expressing HEK cells were mixed and then incubated for 10, 30 or 60 min, Figure 4, Figure 1—figure supplement 1. No higher band corresponding with the full-length NaV1.4 was observed at these time points. Thus, the biochemical data demonstrate that the NaV1.4 DI-II and DIII-IV ERret-intein constructs are competent in HEK cells where they are ligated to produce full-length sodium channels. Figure 4 with 1 supplement see all Download asset Open asset ERret-intein construction allows for expression of functional split WT Nav1.4. (A) Schematic of Nav1.4 protein with ERret-intein sequence engineered on the C- (blue) and N-terminus (orange) of domains I-II and III-IV, respectively. (B) Western blot shows reconstitution and covalent linking of split-intein Nav1.4 proteins following expression in HEK293 cells. Lane 1, full-length WT Nav1.4; Lane 2, front half WT Nav1.4 I-II N-intein; Lane 3, Back half WT Nav1.4 III-IV C-intein; Lane 4 front half WT Nav1.4 I-II N-intein + back half WT Nav1.4 III-IV C-intein expressed at 1:1; Lane 5, front half WT Nav1.4 I-II N-intein + back half WT Nav1.4 III-IV C-intein expressed at 2:1, respectively. (C) Representative Nav1.4 currents 12 hr following co-injection of front half WT Nav1.4 I-II N-intein + back half WT Nav1.4 III-IV C-intein. (D) Quantification of peak Na+ current @ −20 mV following injection of full-length WT Nav1.4 (circles; n = 10), front half WT Nav1.4 I-II N-intein (triangles; n = 6), back half WT Nav1.4 III-IV C-intein (squares; n = 7) and both front half WT Nav1.4 I-II N-intein + back half WT Nav1.4 III-IV C-intein (squares; n = 10). https://doi.org/10.7554/eLife.18976.008 Finally, we aimed to see if the split-intein ERret strategy could assist with nonsense suppression efficiency when attempting to simultaneously suppress two stop sites within a single cRNA transcript. The data in Figure 3 with Shaker ERret-inteins show that this approach can be used to produce tetrameric K+ channel complexes containing two ncAA, but these are within the dimer of dimers K+ channel structural context (i.e. not within the same polypeptide). In contrast, double suppression within the same reading frame poses a particular challenge. Specifically, the ability of an acylated tRNA in the oocyte expression system to suppress stop sites has variable efficiency that is compounded when two stop sites are present, resulting in the expression of full-length channels that are far below that of WT channels, and often immeasurable using electrophysiological methods. Thus, suppression at the second site down-stream from the first compounds the overall efficiency of producing a full-length peptide. We hypothesized that if the two sites are suppressed independently, within the individual Nav1.4 I-II and III-IV hemi-channels for instance, and then subsequently ligated via the ERret-intein sequences that the reduced expression would not be compounded but only limited by the site with the lower suppression efficiency. To test this possibility directly, stop (TAG) sites were engineered into the DI-II construct at Tyr401 near the outer vestibule of the selectivity filter and within the DIII-IV hemi-channel at Phe1304, a site within the DIII-IV linker. When expressed as hemi-channels, the simultaneous dual suppression with Phe-tRNA at Tyr401 and Phe1304 yielded macroscopic sodium currents with gating similar to wildtype NaV1.4 demonstrating that the split ERret-intein method enables dual suppression, Table 1, Figure 5—figure supplement 1. The incorporation was specific to encoding of a Phe residue, because co-injecting the NaV1.4 TAG cRNA with a tRNA lacking an acylated amino acid failed to produce sodium current, Figure 5. Figure 5 with 1 supplement see all Download asset Open asset ERret-intein construction allows for expression of functional split Nav1.4 with two suppressed codons. (A) Co-injection of 25 ng of cRNA encoding Nav1.4 I-II N-intein Tyr401TAG + Nav1.4 III-IV C-intein WT + tRNA(Phe) (left), Nav1.4 I-II N-intein WT + Nav1.4 III-IV C-intein F1304TAG + tRNA(Phe) (center) and Nav1.4 I-II N-intein Tyr401TAG + Nav1.4 III-IV C-intein Phe1304TAG + tRNA(Phe) (right), resulted in robust currents at 24 hr (lower panels). (B) Quantification of peak Na+ current @ −20 mV following injection of 25 ng of cRNA of constructs in A with tRNA(Phe) (open symbols) and uncharged tRNA (closed symbols). (C) Representative Nav1.4 current following co-injection of 50 ng of cRNA encoding Nav1.4 I-II N-intein Y401TAG and Nav1.4 III-IV C-intein Phe1304TAG constructs. (D) Quantification of peak Na+ current @ −20 mV following injection of 50 ng of cRNA encoding Nav1.4 I-II N-intein Tyr401TAG and Nav1.4 III-IV C-intein Phe1304TAG (orange) constructs with tRNA(Phe) (open symbols) and uncharged tRNA (closed symbols). https://doi.org/10.7554/eLife.18976.010 Discussion Taken together, the results presented here advance the specific molecular understanding of C-type inactivation and introduces a novel approach which employs the use ncAAs with split intein protein engineering. MD simulations indicate that the Trp434Ind substitution alters the orientation of the hydrogen bond partner, Asp447. Notably, the ‘flipped out’ orientation of Asp447 occurs in WT channels, but does so briefly and returns to the hydrogen-bonded state. Thus, the simulations suggest that the Ind substitution increases the likelihood of the ‘flipped out’ Asp447 conformation, which disrupts the local environment of the selectivity filter. This orientation of Asp447 allows water to fill the space behind the selectivity filter and simultaneously alters the local electrostatics properties, two outcomes that likely impact the occupancy of potassium ions in the filter. One possibility is that the reorientation of Asp447 may represent an event in the mechanism of C-type inactivation as the channel transitions from the open/conductive (O/C) to a C-type inactivated conformation (O/I), see Panyi and Deutsch, 2007. Further, the breakage of two versus one of the four Asp447-Trp434 hydrogen bonds further accelerates C-type inactivation, possibly due to the loss of an additional hydrogen bond and ultimately results in a concomitant destabilization of the open pore. However, the data do not inform on the details of movements required to break the hydrogen bonds or the nature of the final non-conducting state, an event that occurs downstream from hydrogen bond breakage. We previously attempted to understand domain-specific contributions of hydrogen bonding and C-type inactivation with a Shaker concatemer with all four subunits tethered into a single peptide (Yang et al., 1997). However, while this construct tolerated a single Trp434Phe mutation by site-directed mutagenesis (Pless et. al, 2013, eLife) encoding an ncAA was not feasible as the concatemer displayed a significant decrease in overall expression, a common effect of protein concatenation. Here we describe a protein engineering approach for altering side-chain chemistry prior to concatenation that produces macroscopic potassium currents from Shaker channels containing two Ind amino acids. Split intein sequences have been used to ligate a variety of protein fragments (Shah and Muir, 2014) and the ERret motif used in this study has been shown to work in several types of eukaryotic cells including yeast (Zerangue et al., 1999). Therefore, this approach could be used to examine other proteins in other cellular environments for studying the effects of dominant negative mutations, heteromers, etc. Western blot data of expressed Nav1.4 ERret-intein hemi-channels demonstrates the effectiveness of the approach outside of the Xenopus laevis oocyte expression system and also suggests the ERret-intein can be transferred to other channel types aside from potassium channels. Encoding of multiple ncAA within an individual protein complex remains a significant challenge. Where success is reported, it stems primarily from improving the efficiency of the suppression process and tRNA delivery (Neumann et al., 2010; Wan et al., 2010; Johnson et al., 2011; Chatterjee et al., 2013; Xiao et al., 2013; Lammers et al., 2014). In contrast, the approach described here changes the nature of the dual suppression problem by rescuing each transcript independently prior to protein ligation thus abrogating the loss of expression due to the compounded efficiency of multiple suppression events. Further, the sodium channel data obtained with dual TAG sites suggests that the approach will be useful for suppression of multiple stop sites, a possibility that will be valuable for encoded FRET, once such probes become available. Materials and methods Molecular dynamics simulations Request a detailed protocol Initial coordinates for the molecular dynamic simulations were modeled based on the crystal structure of Kv1.2/Kv2.1 Voltage-gated potassium channel chimera (PDB ID:2R9R). Considering the high sequence identity (87%) of the pore domain between Kv1.2 and Shaker, several non-conserved residues were mutated from Kv1.2 to Shaker, and all of the conserved residue side-chains still maint
