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Structure/Function Studies of the ?4 Subunit Reveal Evolutionary Loss of a GlyR Subtype Involved in Startle and Escape Responses.

ABSTRACT: Inhibitory glycine receptors (GlyRs) are pentameric ligand-gated anion channels with major roles in startle disease/hyperekplexia (GlyR ?1), cortical neuronal migration/autism spectrum disorder (GlyR ?2), and inflammatory pain sensitization/rhythmic breathing (GlyR ?3). However, the role of the GlyR ?4 subunit has remained enigmatic, because the corresponding human gene (GLRA4) is thought to be a pseudogene due to an in-frame stop codon at position 390 within the fourth membrane-spanning domain (M4). Despite this, a recent genetic study has implicated GLRA4 in intellectual disability, behavioral problems and craniofacial anomalies. Analyzing data from sequenced genomes, we found that GlyR ?4 subunit genes are predicted to be intact and functional in the majority of vertebrate species-with the exception of humans. Cloning of human GlyR ?4 cDNAs excluded alternative splicing and RNA editing as mechanisms for restoring a full-length GlyR ?4 subunit. Moreover, artificial restoration of the missing conserved arginine (R390) in the human cDNA was not sufficient to restore GlyR ?4 function. Further bioinformatic and mutagenesis analysis revealed an additional damaging substitution at K59 that ablates human GlyR ?4 function, which is not present in other vertebrate GlyR ?4 sequences. The substitutions K59 and X390 were also present in the genome of an ancient Denisovan individual, indicating that GLRA4 has been a pseudogene for at least 30,000-50,000 years. In artificial synapses, we found that both mouse and gorilla ?4? GlyRs mediate synaptic currents with unusually slow decay kinetics. Lastly, to gain insights into the biological role of GlyR ?4 function, we studied the duplicated genes glra4a and glra4b in zebrafish. While glra4b expression is restricted to the retina, using a novel tol2-GAL4FF gene trap line (SAIGFF16B), we found that the zebrafish GlyR ?4a subunit gene (glra4a) is strongly expressed in spinal cord and hindbrain commissural neurones. Using gene knockdown and a dominant-negative GlyR ?4aR278Q mutant, we found that GlyR ?4a contributes to touch-evoked escape behaviors in zebrafish. Thus, although GlyR ?4 is unlikely to be involved in human startle responses or disease states, this subtype may contribute to escape behaviors in other organisms.

SUBMITTER: Leacock S

PROVIDER: S-EPMC5797729 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Structure/Function Studies of the α4 Subunit Reveal Evolutionary Loss of a GlyR Subtype Involved in Startle and Escape Responses.

Leacock Sophie S Syed Parnayan P James Victoria M VM Bode Anna A Kawakami Koichi K Keramidas Angelo A Suster Maximiliano M Lynch Joseph W JW Harvey Robert J RJ

Frontiers in molecular neuroscience 20180131

Inhibitory glycine receptors (GlyRs) are pentameric ligand-gated anion channels with major roles in startle disease/hyperekplexia (GlyR α1), cortical neuronal migration/autism spectrum disorder (GlyR α2), and inflammatory pain sensitization/rhythmic breathing (GlyR α3). However, the role of the GlyR α4 subunit has remained enigmatic, because the corresponding human gene (<i>GLRA4</i>) is thought to be a pseudogene due to an in-frame stop codon at position 390 within the fourth membrane-spanning ...[more]

PMID: 29445326

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Project description:Glycine receptors (GlyRs) containing the α2 subunit govern cell fate, neuronal migration and synaptogenesis in the developing cortex and spinal cord. Rare missense variants and microdeletions in the X-linked GlyR α2 subunit gene (GLRA2) have been associated with human autism spectrum disorder (ASD), where they typically cause a loss-of-function via protein truncation, reduced cell-surface trafficking and/or reduced glycine sensitivity (e.g., GLRA2Δex8-9 and extracellular domain variants p.N109S and p.R126Q). However, the GlyR α2 missense variant p.R323L in the intracellular M3-M4 domain results in a gain-of-function characterized by slower synaptic decay times, longer duration active periods and increases in channel conductance. This study reports the functional characterization of four missense variants in GLRA2 associated with ASD or developmental disorders (p.V-22L, p.N38K, p.K213E, p.T269M) using a combination of bioinformatics, molecular dynamics simulations, cellular models of GlyR trafficking and electrophysiology in artificial synapses. The GlyR α2V-22L variant resulted in altered predicted signal peptide cleavage and a reduction in cell-surface expression, suggestive of a partial loss-of-function. Similarly, GlyR α2N38K homomers showed reduced cell-surface expression, a reduced affinity for glycine and a reduced magnitude of IPSCs in artificial synapses. By contrast, GlyR α2K213E homomers showed a slight reduction in cell-surface expression, but IPSCs were larger, with faster rise/decay times, suggesting a gain-of-function. Lastly, GlyR α2T269M homomers exhibited a high glycine sensitivity accompanied by a substantial leak current, suggestive of an altered function that could dramatically enhance glycinergic signaling. These results may explain the heterogeneity of clinical phenotypes associated with GLRA2 mutations and reveal that missense variants can result in a loss, gain or alteration of GlyR α2 function. In turn, these GlyR α2 missense variants are likely to either negatively or positively deregulate cortical progenitor homeostasis and neuronal migration in the developing brain, leading to changes in cognition, learning, and memory.

Project description:Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (α4)2(β2)3) or low-sensitivity (LS) (α4)3(β2)2) isoforms of human α4β2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using (86)Rb(+) efflux in a stably transfected SH-EP1-hα4β2 human epithelial cell line, and two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS α4β2-nAChR constructs (HSP and LSP). Unlike previously studied agonists, desensitization by the highly selective agonists A-85380 [3-(2(S)-azetidinylmethoxy)pyridine] and sazetidine-A (Saz-A) preferentially reduced α4β2-nAChR HS-phase versus LS-phase responses. The concatenated-nAChR experiments confirmed that approximately 20% of LS-isoform acetylcholine-induced function occurs in an HS-like phase, which is abolished by Saz-A preincubation. Six mutant LSPs were generated, each targeting a conserved agonist binding residue within the LS-isoform-only α4(+)/(-)α4 interface agonist binding site. Every mutation reduced the percentage of LS-phase function, demonstrating that this site underpins LS-phase function. Oocyte-surface expression of the HSP and each of the LSP constructs was statistically indistinguishable, as measured using β2-subunit-specific [(125)I]mAb295 labeling. However, maximum function is approximately five times greater on a "per-receptor" basis for unmodified LSP versus HSP α4β2-nAChRs. Thus, recruitment of the α4(+)/(-)α4 site at higher agonist concentrations appears to augment otherwise-similar function mediated by the pair of α4(+)/(-)β2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two α4β2-nAChR isoforms, and demonstrate that HS versus LS α4β2-nAChR activity can be selectively manipulated using pharmacological approaches. Since α4β2 nAChRs are the predominant neuronal subtype, these discoveries likely have significant functional implications, and may provide important insights for drug discovery and development.

Dataset Information

Structure/Function Studies of the ?4 Subunit Reveal Evolutionary Loss of a GlyR Subtype Involved in Startle and Escape Responses.

Publications

Structure/Function Studies of the α4 Subunit Reveal Evolutionary Loss of a GlyR Subtype Involved in Startle and Escape Responses.

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