Biology subjects

Couch, T.

Publications and source records attributed to Couch, T..

7 recordsLinked to original sources

bioRxiv2025

Anticodon Edited Transfer RNAs (ACE-tRNAs) Encoded as Therapeutic Nonviral Minimal DNA Vectors

Nonsense mutations, resulting from a premature termination codon (PTC), make up [~]11% of all genetic lesions causing disease, affecting millions of people worldwide. Nonsense suppressor anticodon-edited tRNAs (ACE-tRNAs) have emerged as a therapeutic modality for the rescue of PTCs. Delivery of ACE-tRNAs in vivo has been achieved by adeno-associated viral vector and RNA-lipid nanoparticle, however due to drawbacks associated with these approaches, DNA delivery remains an attractive approach. DNA-based approaches afford ease of manufacturing at a relatively low cost and exhibit improved therapeutic durability and safety as compared to viral vector-or RNA-based approaches. Due to the small size of human tRNA genes employed as ACE-tRNAs, in principle, DNA vectors <200 base pairs (bp) in size (minivectors) could be utilized for delivery of actively transcribed ACE-tRNAs. Here we demonstrate that linear DNA ACE-tRNA vectors as small as 200 bp effectively suppress several nonsense mutations in CFTR and REP1, and that ACE-tRNA minivectors display significantly improved bioavailability, reduced innate immune burden, and superior biostability as compared to conventional plasmid DNA vectors.

molecular biology↗

bioRxiv2024

The impact of subunit type, alternative splicing, and auxiliary proteins on AMPA receptor trafficking

AMPA receptors underlie fast excitatory synaptic transmission in the mammalian nervous system and are critical for the expression of synaptic plasticity. Four genes encode the AMPA subunits, each subject to RNA editing and alternative splicing at multiple positions. In addition, each tetrameric AMPA receptor can harbor up to four auxiliary proteins, of which there are multiple types. Subunit type, alternative splicing, and auxiliary proteins are all known to affect AMPA receptor gating and trafficking. However, determining which factors dominate AMPA receptor trafficking requires high throughput assessment of trafficking across multiple conditions. Here, we deploy two such methods to assess the relative contribution of AMPA subunit type (GluA1 versus GluA2), alternative splicing (flip versus flop), and various transmembrane AMPA receptor regulatory proteins (TARPs) to AMPA receptor trafficking. We find that subunit type is the most important factor, with GluA2 showing much better surface expression than GluA1, and alternative splicing plays a secondary role, with flip subunits consistently outperforming flop variants in surface expression across all conditions. Type 1 TARPs ({gamma}2-4 and {gamma}8) enhance surface trafficking, while Type 2 TARPs ({gamma}5 and {gamma}7) reduced surface expression, although we could not detect differences within each type. These data will be a helpful resource in comparing surface expression across a variety of AMPA receptor compositions. Our assays will also enable high throughput assessment of novel disease-associated mutations, chimeras, and auxiliary and chaperone proteins.

neuroscience↗

bioRxiv2024

Unique Pharmacology of mGluR homo- and heterodimers

Background and PurposeMetabotropic glutamate receptors (mGlus) are obligate dimer G protein coupled receptors that can all homodimerize and heterodimerize in select combinations. Responses of mGlu heterodimers to selective ligands, including orthosteric agonists and allosteric modulators, are largely unknown. Experimental ApproachThe pharmacological properties of each group II and III mGlu homodimer (except mGlu6) and several heterodimers were examined when stochastically assembled in HEK293T cells, or specifically measured using an improved G protein mediated BRET assay employing complimented fragments of NanoLuciferase. ResultsStochastically assembled receptors adopted unique signaling characteristics. Some favored the potency, efficacy or signaling kinetics of a dominant subunit, while others exhibited blended profiles reflective of a combination of homo- and heterodimers at various ratios of expressed receptor. Finally, group II and III mGlu dimers were examined for responses to selective agonists and allosteric modulators. Effects of glutamate and selective group II and III orthosteric agonists were found to result in unique concentration response profiles when examining each combination of group II and II mGlu. Effects of select allosteric modulators were examined for each mGlu2 containing dimer as well as several group III dimer pairs. Likewise, allosteric modulator effects were often unique across dimers containing the targeted subunit of the ligand being tested. ConclusionsResults demonstrate that mGlu dimers respond uniquely to selective ligands, and show that the mGlu family is not governed by generalizable rules dictating consequences of dimeric subunit interactions leading to signaling consequences.

pharmacology and toxicology↗

bioRxiv2024

Anticodon Engineered Transfer RNAs (ACE-tRNAs) are a Platform Technology for Suppressing Nonsense Mutations

Nonsense mutations arise from single nucleotide substitutions that result in premature termination codons (PTCs). PTCs result in little to no full-length protein production and loss of mRNA expression through the nonsense-mediated mRNA decay (NMD) pathway. We demonstrate that anticodon engineered (ACE-) tRNAs efficiently suppress the most prevalent cystic fibrosis (CF) causing PTCs, promoting significant rescue of endogenous cystic fibrosis transmembrane conductance regulator (CFTR) transcript abundance and channel function in different model systems. We demonstrate that our best-performing ACE-tRNA, that decodes all UGA PTCs to a leucine amino acid, markedly rescues CFTR channel function from the most prevalent CF causing PTCs that arise from non-leucine encoding codons. Using this single ACE-tRNA variant, we demonstrate significant rescue of CFTR channel function in an immortalized airway cell line and two different primary CF patient-derived intestinal cell models with CFTR nonsense mutations. Thus, ACE-tRNAs have promise as a platform therapeutic for CF and other nonsense-associated diseases.

molecular biology↗

bioRxiv2024

Proline substitutions in the ASIC1 β11-12 linker slow desensitization

Desensitization is a prominent feature of nearly all ligand gated ion channels. Acid-sensing ion channels (ASIC) undergo desensitization within hundreds of milliseconds to seconds upon continual extracellular acidification. The ASIC mechanism of desensitization is primarily due to the isomerization or "flipping" of a short linker joining the 11th and 12th beta sheets in the extracellular domain. In the resting and active states this {beta}11-12 linker adopts an "upward" conformation while in the desensitized conformation the linker assumes a "downward" state. To accommodate this "downward" state, specific peptide bonds within the linker adopt either trans-like or cis-like conformations. Since proline-containing peptide bonds undergo cis-trans isomerization very slowly, we hypothesized that introducing proline residues in the linker may slow or even abolish ASIC desensitization, potentially providing a valuable research tools. Proline substitutions in the chicken ASIC1 {beta}11-12 linker (L414P and Y416P) slowed desensitization decays approximately 100 to 1000-fold as measured in excised patches. Both L414P and Y416P shifted the steady state desensitization curves to more acidic pHs while activation curves and ion selectivity of these slow-desensitizing currents were largely unaffected. To investigate the functional stoichiometry of desensitization in the trimeric ASIC, we created families of L414P and Y416P concatemers with zero, one, two or three proline substitutions in all possible configurations. Introducing one or two L414P or Y416P mutations only slightly attenuated desensitization, suggesting that conformational changes in the remaining faster wild type subunits were sufficient to desensitize the channel. These data highlight the unusual cis-trans isomerization mechanism of ASIC desensitization and support a model where a single subunit is sufficient to desensitize the entire channel.

biophysics↗

bioRxiv2021

Topography and motion of the acid-sensing ion channel intracellular domains

Acid-sensing ion channels (ASICs) are trimeric cation-selective channels activated by decreases in extracellular pH. The intracellular N and C terminal tails of ASIC1 influence channel gating, trafficking, and signaling in ischemic cell death. Despite several x-ray and cryo-EM structures of the extracellular and transmembrane segments of ASIC1, these important intracellular tails remain unresolved. Here we describe the coarse topography of the cASIC1 intracellular domains determined by FRET, measured using either fluorescent lifetime imaging or patch clamp fluorometry. We find the C terminal tail projects into the cytosol by approximately 35 [A] and that the N and proximal segment of the C tail from the same subunits are closer than adjacent subunits. Using pH-insensitive fluorescent proteins, we fail to detect any relative movement between the N and C tails upon extracellular acidification but do observe axial motions of the membrane proximal segments towards the plasma membrane. Taken together, our study furnishes a coarse topographic map of the ASIC intracellular domains while providing directionality and context to intracellular conformational changes induced by extracellular acidification.

physiology↗

bioRxiv2020

Mutation of a conserved Gln residue does not abolish desensitization of acid-sensing ion channel 1

Desensitization is a common feature of ligand-gated ion channels although the molecular cause varies widely between channel types. Mutations that substantially reduce or abolish desensitization have been described for many ligand-gated ion channels including glutamate, GABA, glycine and nicotinic receptors but not for acid-sensing ion channels (ASICs) until recently. Mutating Gln276 to a glycine in human ASIC1a was reported to mostly abolish desensitization at both the macroscopic and single channel levels, potentially providing a valuable tool for subsequent studies. However, we find that in both human and chicken ASIC1 the effect of Q276G is modest. In chicken ASIC1, the equivalent Q277G slightly reduces desensitization when using pH 6.5 as a stimulus but desensitizes essentially like wild type when using more acidic pH values. In addition, steady-state desensitization is intact, albeit right-shifted, and recovery from desensitization is accelerated. Molecular dynamics simulations indicate that the Gln277 side chain participates in a hydrogen bond network that might stabilize the desensitized conformation. Consistent with this, destabilizing this network with the Q277N or Q277L mutations largely mimics the Q277G phenotype. In human ASIC1a, Q276G does not substantially reduce desensitization but surprisingly slows entry to and exit from the desensitized state, thus requiring longer agonist applications to reach equilibrium. Our data reveal that while the Q/G mutation does not substantially impair desensitization as previously reported, it does point to unexpected differences between chicken and human ASICs and the need for careful scrutiny before using this mutation in future studies.

biophysics↗