Structure/Function Studies of the alpha 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 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.Leacock, Sophie Syed, Parnayan James, Victoria M. Bode, Anna Kawakami, Koichi Keramidas, Angelo Suster, Maximiliano Lynch, Joseph W. Harvey, Robert J. <br />