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

Inhibitory glycine receptors (GlyRs) are pentameric ligand-gated anion channels with major roles in startle disease/hyperekplexia (GlyR alpha 1), cortical neuronal migration/autism spectrum disorder (GlyR alpha 2), and inflammatory pain sensitization/rhythmic breathing (GlyR alpha 3). However, the role of the GlyR alpha 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 alpha 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 alpha 4 cDNAs excluded alternative splicing and RNA editing as mechanisms for restoring a full-length GlyR alpha 4 subunit. Moreover, artificial restoration of the missing conserved arginine (R390) in the human cDNA was not sufficient to restore GlyR alpha 4 function. Further bioinformatic and mutagenesis analysis revealed an additional damaging substitution at K59 that ablates human GlyR alpha 4 function, which is not present in other vertebrate GlyR alpha 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 alpha 4 beta GlyRs mediate synaptic currents with unusually slow decay kinetics. Lastly, to gain insights into the biological role of GlyR alpha 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 alpha 4a subunit gene (glra4a) is strongly expressed in spinal cord and hindbrain commissural neurones. Using gene knockdown and a dominant-negative GlyR alpha 4a(R278Q) mutant, we found that GlyR alpha 4a contributes to touch-evoked escape behaviors in zebrafish. Thus, although GlyR alpha 4 is unlikely to be involved in human startle responses or disease states, this subtype may contribute to escape behaviors in other organisms.