Premature termination codons (PTCs) are a major class of pathogenic variants that underlie rare inherited disorders, including forms of childhood blindness. Therapeutic suppression of these “nonsense mutations” offers a gene- and position-agnostic strategy to restore protein function. Our previous work established that the W53X PTC in the KCNJ13 gene causes Leber congenital amaurosis type 16 (LCA16) by disrupting the inwardly rectifying potassium channel Kir7.1, leading to retinal pigment epithelium (RPE) dysfunction. Here, we present a proof-of-concept approach using anticodon-engineered transfer RNA (ACE-tRNA) to promote targeted translational readthrough. We engineered a suppressor tRNA encoding tryptophan (ACE-tRNATrp.UAG) to selectively recognize the UAG stop codon at the W53X site, enabling incorporation of the correct amino acid and restoration of full-length Kir7.1 protein. Delivery of ACE-tRNA via helper-dependent adenovirus (HDAd) resulted in robust rescue of channel function in heterologous systems expressing mutant KCNJ13 and in patient-derived human induced pluripotent stem cell (hiPSC)-RPE cells. Functional recovery was confirmed by electrophysiological assays demonstrating restored inwardly rectifying currents and membrane potential. Importantly, subretinal delivery of HDAd-ACE-tRNATrp.UAG in a W53X mouse model led to partial restoration of RPE physiology, as measured by electroretinography, without evidence of retinal toxicity. Together, these findings establish ACE-tRNA-mediated suppression as a viable therapeutic strategy for nonsense mutations in multimeric ion channels and provide a translational framework for precision treatment of inherited retinal diseases.

Kv7.1 is a cardiac voltage-gated potassium channel that underlies the delayed rectifier current (IKS) in the heart. The slow response to membrane depolarization is a hallmark feature of this channel's physiology, yet the mechanistic basis of how voltage promotes the open potassium conducting state is unknown. We focused on previously identified aromatic residues which might couple the pore and voltage-sensing domains (VSDs) by using a chemical tuning approach whereby aromatic residues are modified by serial fluorination. The data show that serial fluorination at one site (F232 on the S4 helix, within the VSD) resulted in a stepwise voltage-gating shift, where each added fluorine atom further biased channel opening to more negative voltages. Mutant-cycle analysis of proximal positively charged amino acids indicates that F232 likely forms a cation-π interaction with K285, a residue at the tip of the S5 segment in the pore domain. Using cryoelectron microscopy, a partial structure of the F232 penta-F-Phe Kv7.1 (KCNQ1) open channel was resolved to 6 Å. The data support a gating mechanism whereby the F232-K285 cation-π interaction represents an intermediate activated state that is broken prior to channel opening.

Pentameric ligand-gated ion channels (pLGICs) mediate fast inhibitory neurotransmission critical for neuronal network stability. A tyrosine residue in the M2-M3 linker of inhibitory pLGICs, conserved for over 600 million years, is positioned where it could hydrogen bond (H-bond) to the backbone of the neighboring Cys-loop. Given the pathogenic effects of variants of this tyrosine, we hypothesized that this H-bond stabilizes extracellular-to-transmembrane domain coupling essential for channel gating. To test this hypothesis, we used site-directed mutagenesis, noncanonical amino acid incorporation, and electrophysiological recordings in Xenopus laevis oocytes to disrupt this hydrogen bond in GABAA and glycine receptors. Loss of this interaction via tyrosine substitutions or backbone amide modifications that ablate the acceptor or donor, respectively, markedly decrease agonist sensitivity and maximal channel activation, with effects localized to specific subunits. Molecular dynamics simulations support a role for this H-bond in channel gating. These findings reveal a critical atomic interaction underlying a shared mechanism for inhibitory receptor gating and provide a mechanistic explanation for disease-associated mutations linked to epilepsy, neurodevelopmental disability, and hyperekplexia.

ABSTRACT Protein truncating variants caused by UGA stop codons are the most prevalent class of rare variant mutations in neurodevelopmental diseases. Suppressor transfer RNA (sup-tRNA) have therapeutic potential for premature termination codon (PTC) repair, but have thus far underperformed by traditional AAV delivery platforms and progress has been hampered by the lack of methods to non-invasively assess in vivo activity in mammalian brain. To fill this material gap, we utilize transcranial in vivo bioluminescence imaging data from a luciferase-UGA mouse model to enable payload optimization. These data demonstrate that U6 promotor and AAV2/9 capsids have the lowest in vivo activity, whereas self-complementary AAV2/9 with the tRNA in a minimal 100bp genomic context provide broad and efficacious PTC rescue. Further, payload tRNA multiplexing and use of tRNA introns enable efficacy of low viral titers and sustained rescue. tRNA sequencing of scAAV delivered Arg UGA sup-tRNA in brain demonstrate no effects on endogenous tRNA levels, their acylation or processing, and these features are also maintained in scAAV delivered Arg UGA sup-tRNA. Collectively, this work defines a scalable strategy for precision UGA stop codon suppression, supporting development of durable genetic rescue therapies for neurodevelopmental disorders in the mammalian brain. GRAPHICAL ABSTRACT

Protein-truncating variants caused by stop codons are the most prevalent class of rare variant mutations in neurodevelopmental diseases, with UGA codons being most common. Suppressor transfer RNA (sup-tRNA) has therapeutic potential for premature termination codon (PTC) rescue but has thus far underperformed by traditional AAV delivery platforms, and progress has been hampered by the lack of methods to non-invasively assess in vivo activity in mammalian brain. To fill this material gap, we utilize transcranial in vivo bioluminescence imaging data from a luciferase-UGA mouse model to optimize viral payloads with sup-tRNA genes. These data demonstrate that U6 promoter-driven and single-stranded AAV2/9 constructs show variable and dose-dependent activity, whereas self-complementary AAV2/9 with the tRNA in a minimal 100-bp genomic context provides broad and efficacious PTC rescue. Further, payload tRNA multiplexing and use of tRNA introns enable efficacy of low viral titers and sustained rescue. tRNA sequencing of scAAV-delivered ArgUCA sup-tRNA in brain demonstrates no effects on endogenous tRNA levels, their acylation, or processing, and these features are also maintained in the delivered ArgUCA sup-tRNA. Collectively, this work defines a scalable strategy for precision UGA PTC stop codon suppression, supporting development of durable genetic rescue therapies for neurodevelopmental disorders in the mammalian brain.

BACKGROUND:NaV (voltage-gated sodium) channels drive cardiac excitability. Although NaV1.5 is the primary cardiac isoform, the composition and functional contributions of non-NaV1.5 isoforms in the heart remain unclear. METHODS:Here, we developed a chemical-genetic mouse model (NaV1.5GX/GX) in which NaV1.5 can be selectively and reversibly inhibited by acyl- and aryl-sulfonamide compounds (GX [acyl- and aryl-sulfonamide compounds typically denoted by the name GX-### and associated items] drugs). Cardiac activity was assessed by electrocardiograms in vivo, and optical mapping was used for imaging of ex vivo hearts. Whole-cell voltage-clamp in tandem with validated toxins and isoform-selective inhibitors were used to examine sodium current composition. RESULTS:NaV1.5GX/GX mice exhibited normal cardiac function at baseline, but acute GX drug administration caused profound conduction defects and arrhythmias. Whole-heart optical mapping revealed dose-dependent chamber-specific sensitivity to NaV1.5 inhibition, with the right ventricle being the most sensitive, followed by the left ventricle, left atrium, and right atrium. Patch-clamp recordings of isolated cardiomyocytes with application of NaV isoform-selective inhibitors showed that NaV1.5 contributed 93% of sodium current in the left ventricle, 79% in the right ventricle, and 78% in the atria. Non-NaV1.5 isoforms were differentially enriched across chambers: NaV1.8 in the left ventricle, NaV1.1/1.3 in the right ventricle, and NaV1.2/1.6/1.7 in the atria. CONCLUSIONS:These results reveal a surprising chamber-specific isoform landscape of cardiac sodium currents, which may underlie the right ventricular predominant phenotype of Brugada syndrome. These data highlight non-NaV1.5 isoforms as potential mediators of chamber-specific cardiac pathologies and as pharmacological targets.

SCN2A encodes the voltage-gated sodium channel NaV1.2, a central regulator of action potential initiation and propagation in glutamatergic neurons, and one of the strongest single-gene risk factors for autism spectrum disorder. Premature termination codons in SCN2A are widely considered to produce uniform haploinsufficiency through nonsense-mediated mRNA decay, an assumption that underpins current mechanistic and therapeutic models. We generated two mouse lines carrying patient-derived mutations - Scn2aY84X/+ (p.Tyr84UAA; early coding sequence) and Scn2aR1627X/+ (p.Arg1627UGA; terminal coding exon). We assessed allele-specific mRNA expression, NaV1.2 protein expression, ex vivo whole-cell recordings, and behavioral phenotypes in these mice. Allele-specific RNA handling diverged by position: mRNA carrying Y84X engaged partial nonsense-mediated decay, whereas R1627X transcripts were at allelic balance. Despite this difference in RNA fate, NaV1.2 protein was comparably reduced in both lines. Both variants slowed the action potential upstroke, with a larger decrement in Scn2aY84X/+. Spike threshold was depolarized only in Scn2aY84X/+. Mutant neurons showed reduced firing near rheobase. Both lines exhibited increased grooming, but Scn2aY84X/+ alone showed greater exploration and a male-predominant rotarod learning deficit. Locomotion, sociability, and sensorimotor gating were preserved. In maximal electroshock testing, mortality was reduced in both lines without changes in seizure threshold or severity. Our results show that SCN2A premature termination codon position determines allele-specific effects on neuronal excitability and behavior, where both NMD and phenotypes of the Scn2aY84X/+ line are more penetrant. These data challenge the assumption of uniform haploinsufficiency and directly support allele-tailored mechanistic studies and therapeutic strategies.

Premature termination codons (PTCs) are associated with rare genetic disorders. Inducing targeted read-through of these 'nonsense mutations' presents a potential therapeutic strategy for modifying disease outcomes. We previously reported that one such PTC, W53X, in the KCNJ13 gene causes blindness and Leber congenital amaurosis type-16 (LCA-16) due to loss of function of the inwardly rectifying potassium channel 7.1 (Kir7.1). Here, we present the proof of concept of a therapeutic approach based on anticodon-engineered transfer RNA (ACE-tRNA). The ACE-tRNA encodes the amino acid tryptophan (Trp) and suppresses the W53X PTC, restoring full-length protein expression. We used helper-dependent adenovirus (HDAd) to deliver the ACE-tRNATrp.UAG (tRNATrp.UAG) and rescue Kir7.1 function and physiology in patient-specific human induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE) cells. Furthermore, in a W53X mouse model of LCA16, HDAd delivery of tRNATrp.UAG resulted in durable restoration of vision as measured by retinography. This study provides the first example of the therapeutic application of ACE-tRNA for treating an inherited form of blindness.

GABAA receptors (GABAARs) are the primary inhibitory neurotransmitter receptors throughout the central nervous system. Genetic mutations causing their dysfunction are related to a broad spectrum of human disorders such as epilepsy, neurodevelopment and intellectual disability, autism spectrum disorder, schizophrenia, and depression. GABAARs are also important drug targets for anxiolytics, anticonvulsants, antidepressants, and anesthetics. Despite significant progress in understanding their three-dimensional structure, a critical gap remains in determining the molecular basis for channel gating. We recently identified mutations in the M2-M3 linkers that suggest linker flexibility has asymmetric subunit-specific correlations with channel opening. Here we use non-canonical amino acids (ncAAs) to investigate the role of main-chain H-hydrogen bonds (H-bonds) that may stabilize the M2-M3 linkers. We show that a single main-chain H-bond within the β2 subunit M2-M3 linker inhibits pore opening and is required to keep the unliganded channel closed. Furthermore, breaking this H-bond accounts for approximately one third of the energy used to open the channel during activation by GABA. In contrast, the analogous H-bond in the α1 subunit has no effect on gating. Our molecular simulations support the idea that channel opening involves the state-dependent breakage/disruption of a specific main-chain H-bond within the β2 subunit M2-M3 linker.

Voltage-gated sodium channels initiate action potentials and control electrical signaling throughout the animal kingdom. Fast inactivation is an essential auto-inhibitory mechanism and requisite component of sodium channel physiology. Recent structural and electrophysiological results are inconsistent with the canonical "ball and chain" model of fast inactivation thus necessitating an updated theoretical framework. Here, we use encoded fluorescence spectroscopy and high-resolution electrophysiology to capture key steps in the fast inactivation mechanism, from voltage-sensor activation to pore occlusion, an ultra-fast process which occurs in less than 2 milliseconds. Upon depolarization, activation of the domain IV voltage sensor initiates cytoplasmic DIII_DIV linker movement and quickly repositions the IFM motif into a hydrophobic pocket adjacent to the pore. This triggers a structural rearrangement of the pocket. The phenylalanine of the IFM motif contacts the pore-forming helices via a hydrophobic interaction with S6 of DIV and an aromatic/hydrophobic interaction with S6 of DIIII. These two interactions occur only after both S6 segments rotate, thus exposing the hydrophobic gate into the pore producing the fast inactivation. Based on the current results, we propose an alternative "lock and key" model to explain the molecular mechanism of fast inactivation.

The KCNJ13 gene encodes the Kir7.1 protein, and mutations cause Leber’s Congenital Amaurosis (LCA) and Snowflake Vitreoretinal Degeneration (SVD), leading to early-onset vision loss. One such nonsense mutation, R166X, results in nystagmus, poor night vision, and visual impairment. We examined therapeutic options for the R166X nonsense point mutation. Unlike missense genetic mutations, which can be rectified by gene augmentation and genome editing, nonsense mutations provide an opportunity to test additional readthrough therapies. Attempts to produce human induced pluripotent stem cells (hiPSCs) with the R166X mutation (CGA to TGA) were unsuccessful. This may be due to challenges in chromatin structure and folding that hinder access to the targeted loci. Therefore, we constructed an open reading frame (ORF) stably integrated HEK293T line by inserting the wild type (WT) or R166X-KCNJ13 gene using the FLP-FRT recombinase technique. While genome editing strategies did not repair the R166X mutation efficiently in this cell line, an arginine anticodon-engineered tRNA (ACE-tRNAArg.UGA) restored K+ channel expression and function. This study provides an example in precision medicine where translational readthrough strategies can rescue channel function at a mutation that is difficult to correct via genome editing.

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.

GABAA receptors are the primary inhibitory neurotransmitter receptors throughout the central nervous system. Despite significant progress understanding their three-dimensional structure, a critical gap remains in determining the molecular basis for channel gating. We recently identified M2-M3 linker mutations that suggest linker flexibility has asymmetric subunit-specific correlations with channel opening. Here we use non-canonical amino acids (ncAAs) to investigate the role of main-chain H-hydrogen bonds (H-bonds) that may stabilize the M2-M3 linkers. We show that a single main-chain H-bond within the β2 subunit M2-M3 linker inhibits pore opening and is required to keep the unliganded channel closed. Furthermore, breaking this H-bond during channel opening accounts for approximately one third of the activation energy derived from GABA binding. In contrast, the analogous H-bond in the α1 subunit has no effect on gating. Our observations suggest that channel opening involves state-dependent breakage/disruption of a specific main-chain H-bond within the β2 subunit M2-M3 linker. ### Competing Interest Statement The authors have declared no competing interest.

Myelin protein zero (MPZ) is a structural transmembrane protein and a major component of peripheral myelin that holds tightly compacted adjacent membrane layers together. The extracellular immunoglobulin-like domain of MPZ (IgMPZ) is thought to adhere apposing membranes together through homomeric interactions; however, the multiple weak interfaces that occur within the IgMPZ crystal structure fail to provide a definitive model for this assembly. Here, equilibrium distributions for IgMPZ oligomeric populations were obtained in solution using NMR and SAXS.

Rapid and effectual inactivation in voltage-gated sodium channels is required for canonical action-potential firing. This "fast" inactivation arises from swift and reversible protein conformational changes that utilize transmembrane segments and the cytoplasmic linker between channel domains III and IV. Until recently, fast inactivation had been accepted to rely on a "ball-and-chain" mechanism whereby a hydrophobic triplet of DIII-IV amino acids (IFM) impairs conductance by binding to a site in central pore of the channel made available by channel opening. New structures of sodium channels have upended this model. Specifically, cryo-electron microscopic structures of eukaryotic sodium channels depict a peripheral binding site for the IFM motif, outside of the pore, opening the possibility of a yet unidentified allosteric mechanism of fast-inactivation gating. We set out to study fast inactivation by photo-trapping human sodium channels in various functional states under voltage control. This was achieved by genetically encoding the crosslinking unnatural amino acid benzophenone phenylalanine at various sites within the DIII-IV linker in the cardiac sodium channel NaV1.5. These data show dynamic state- and positional-dependent trapping of the transient conformations associated with fast inactivation, each yielding different phenotypes and rates of trapping. These data reveal distinct conformational changes that underlie fast inactivation and point to a dynamic environment around the IFM locus.