GlyR α 2, not GlyR α 3, modulates the receptive field surround of OFF retinal ganglion cells

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 (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 α4 subunit genes are predicted to be intact and functional in the majority of vertebrate species—with the exception of humans. Cloning of human GlyR α4 cDNAs excluded alternative splicing and RNA editing as mechanisms for restoring a full-length GlyR α4 subunit. Moreover, artificial restoration of the missing conserved arginine (R390) in the human cDNA was not sufficient to restore GlyR α4 function. Further bioinformatic and mutagenesis analysis revealed an additional damaging substitution at K59 that ablates human GlyR α4 function, which is not present in other vertebrate GlyR α4 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 α4β GlyRs mediate synaptic currents with unusually slow decay kinetics. Lastly, to gain insights into the biological role of GlyR α4 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 α4a subunit gene (glra4a) is strongly expressed in spinal cord and hindbrain commissural neurones. Using gene knockdown and a dominant-negative GlyR α4aR278Q mutant, we found that GlyR α4a contributes to touch-evoked escape behaviors in zebrafish. Thus, although GlyR α4 is unlikely to be involved in human startle responses or disease states, this subtype may contribute to escape behaviors in other organisms.

Section: Introductionmentioning

confidence: 99%

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

Leacock¹,

Syed

James³

et al. 2018

“…GlyR α2 is also pivotal in the modulation of ethanol intake, aversion and preference (Blednov et al, 2015), retinal photoreceptor development (Young and Cepko, 2004) and the control of receptive field surround in retinal ganglion cells (Nobles et al, 2012; Zhang C. et al, 2015). However, more recent studies using a novel Glra2 knockout line provided compelling evidence that extrasynaptic activation of GlyRs containing the α2 subunit in interneurons is vital for control of cortical tangential migration during embryogenesis (Avila et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Structure-Function Analysis of the GlyR α2 Subunit Autism Mutation p.R323L Reveals a Gain-of-Function

Zhang

Harvey³

et al. 2017

Glycine receptors (GlyRs) containing the α2 subunit regulate cortical interneuron migration. Disruption of the GlyR α2 subunit gene (Glra2) in mice leads to disrupted dorsal cortical progenitor homeostasis, leading to a depletion of projection neurons and moderate microcephaly in newborn mice. In humans, rare variants in GLRA2, which is located on the X chromosome, are associated with autism spectrum disorder (ASD) in the hemizygous state in males. These include a microdeletion (GLRA2∆ex8-9) and missense mutations in GLRA2 (p.N109S and p.R126Q) that impair cell-surface expression of GlyR α2, and either abolish or markedly reduce sensitivity to glycine. We report the functional characterization of a third missense variant in GLRA2 (p.R323L), associated with autism, macrocephaly, epilepsy and hypothyroidism in a female proband. Using heterosynapse and macroscopic current recording techniques, we reveal that GlyR α2R323L exhibits reduced glycine sensitivity, but significantly increased inhibitory postsynaptic current (IPSC) rise and decay times. Site-directed mutagenesis revealed that the nature of the amino acid switch at position 323 is critical for impairment of GlyR function. Single-channel recordings revealed that the conductance of α2R323Lβ channels was higher than α2β channels. Longer mean opening durations induced by p.R323L may be due to a change in the gating pathway that enhances the stability of the GlyR open state. The slower synaptic decay times, longer duration active periods and increase in conductance demonstrates that the GlyR α2 p.R323L mutation results in an overall gain of function, and that GlyR α2 mutations can be pathogenic in the heterozygous state in females.

“…Consistent with this, non-synaptic taurine or glycine release onto GlyRs was found to be vital for neocortical and spinal cord development (Flint et al, 1998;Scain et al, 2010). Studies using Glra2 knockout mice have since revealed pivotal roles for GlyR α2 in retinal photoreceptor development (Young and Cepko, 2004) and the control of receptive field surrounds in retinal ganglion cells (Nobles et al, 2012;Zhang et al, 2015a), as well as modulation of ethanol intake, aversion and preference (Blednov et al, 2015;San Martin et al, 2020;Araya et al, 2021). However, a major role for GlyR α2 has been identified in dorsal cortical progenitor homeostasis and cortical interneuron migration (Avila et al, 2013(Avila et al, , 2014.…”

Section: Introductionmentioning

confidence: 71%

Loss, Gain and Altered Function of GlyR α2 Subunit Mutations in Neurodevelopmental Disorders

Chen

Wilson

Schaefer³

et al. 2022

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.