Structure/Function Studies of the α4 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 a1), cortical neuronal migration/autism spectrum disorder (GlyR a2), and inflammatory pain sensitization/rhythmic breathing (GlyR a3). However, the role of the GlyR a4 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 a4 subunit genes are predicted to be intact and functional in the majority of vertebrate species—with the exception of humans. Cloning of human GlyR a4 cDNAs excluded alternative splicing and RNA editing as mechanisms for restoring a full-length GlyR a4 subunit. Moreover, artificial restoration of the missing conserved arginine (R390) in the human cDNA was not sufficient to restore GlyR a4 function. Further bioinformatic and mutagenesis analysis revealed an additional damaging substitution at K59 that ablates human GlyR a4 function, which is not present in other vertebrate GlyR a4 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 a4b GlyRs mediate synaptic currents with unusually slow decay kinetics. Lastly, to gain insights into the biological role of GlyR a4 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 a4a subunit gene (glra4a) is strongly expressed in spinal cord and hindbrain commissural neurones. Using gene knockdown and a dominant-negative GlyR a4aR278Q mutant, we found that GlyR a4a contributes to touch-evoked escape behaviors in zebrafish. Thus, although GlyR a4 is unlikely to be involved in human startle responses or disease states, this subtype may contribute to escape behaviors in other organisms