Abstract
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.
Highlights
Inhibitory glycine receptors (GlyRs) are ligand-gated anion channels, consisting of pentameric combinations of GlyR α and β subunits
Sanger DNA sequencing of 10 human GlyR α4 subunit cDNAs and comparison with the GLRA4 consensus sequence revealed that: (1) All cDNAs encoded a valine at position 57; (2) Two cDNA clones had the change c.1345T >C, p.W421R, suggesting that this could be a common polymorphism in human GLRA4
Given the recent implication of the human GlyR α4 subunit gene in human disease, we sought to: (1) Determine whether the human GlyR α4 subunit is functional or can act in a dominant-negative manner; (2) To characterize the functional properties of α4-subunit GlyRs in artificial synapses; and (3) Determine the biological role of GlyR α4 using zebrafish as a model organism
Summary
Inhibitory glycine receptors (GlyRs) are ligand-gated anion channels, consisting of pentameric combinations of GlyR α and β subunits. GlyR α2 has been shown to control cortical neuronal progenitor homeostasis, migration and circuit formation (Avila et al, 2013, 2014; Morelli et al, 2017) with mild microcephaly (Avila et al, 2014), susceptibility to seizures (Morelli et al, 2017) and deficits in long-term potentiation and object recognition memory (Pilorge et al, 2016) observed in Glra knockout mice Consistent with these findings, loss of function mutations in the human GlyR α2 subunit gene (GLRA2) have been reported in cases of autism spectrum disorder, with additional features in some cases such as language delay and seizures (Pinto et al, 2010; Piton et al, 2011; Iossifov et al, 2014; Pilorge et al, 2016). GlyR α3 is involved in hearing, and has important functions in auditory nerve activity (Dlugaiczyk et al, 2016) and signal-in-noise detection (Tziridis et al, 2017)
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