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 α1), cortical neuronal migration/autism spectrum disorder (GlyR α2), and inflammatory pain sensitization/rhythmic breathing (GlyR α3). However, the role of the GlyR...
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| Language: | English |
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Frontiers Media S.A.
2018-01-01
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| Series: | Frontiers in Molecular Neuroscience |
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| Online Access: | http://journal.frontiersin.org/article/10.3389/fnmol.2018.00023/full |
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| author | Sophie Leacock Parnayan Syed Victoria M. James Anna Bode Koichi Kawakami Angelo Keramidas Maximiliano Suster Joseph W. Lynch Joseph W. Lynch Robert J. Harvey Robert J. Harvey |
| author_facet | Sophie Leacock Parnayan Syed Victoria M. James Anna Bode Koichi Kawakami Angelo Keramidas Maximiliano Suster Joseph W. Lynch Joseph W. Lynch Robert J. Harvey Robert J. Harvey |
| author_sort | Sophie Leacock |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-14T19:59:50Z |
| format | Article |
| id | doaj.art-5eb871dcc3324460af97ccca12370326 |
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| issn | 1662-5099 |
| language | English |
| last_indexed | 2024-12-14T19:59:50Z |
| publishDate | 2018-01-01 |
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| series | Frontiers in Molecular Neuroscience |
| spelling | doaj.art-5eb871dcc3324460af97ccca123703262022-12-21T22:49:12ZengFrontiers Media S.A.Frontiers in Molecular Neuroscience1662-50992018-01-011110.3389/fnmol.2018.00023327470Structure/Function Studies of the α4 Subunit Reveal Evolutionary Loss of a GlyR Subtype Involved in Startle and Escape ResponsesSophie Leacock0Parnayan Syed1Victoria M. James2Anna Bode3Koichi Kawakami4Angelo Keramidas5Maximiliano Suster6Joseph W. Lynch7Joseph W. Lynch8Robert J. Harvey9Robert J. Harvey10Department of Pharmacology, UCL School of Pharmacy, London, United KingdomQueensland Brain Institute, The University of Queensland, Brisbane, QLD, AustraliaDepartment of Pharmacology, UCL School of Pharmacy, London, United KingdomQueensland Brain Institute, The University of Queensland, Brisbane, QLD, AustraliaDivision of Molecular and Developmental Biology, National Institute of Genetics and Department of Genetics, Graduate University for Advanced Studies (SOKENDAI), Mishima, JapanQueensland Brain Institute, The University of Queensland, Brisbane, QLD, AustraliaNeural Circuits and Behaviour Group, Uni Research AS, Bergen, NorwayQueensland Brain Institute, The University of Queensland, Brisbane, QLD, AustraliaSchool of Biomedical Sciences, The University of Queensland, Brisbane, QLD, AustraliaSchool of Health and Sport Sciences, University of the Sunshine Coast, Sippy Downs, QLD, AustraliaSunshine Coast Health Institute, Birtinya, QLD, AustraliaInhibitory 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.http://journal.frontiersin.org/article/10.3389/fnmol.2018.00023/fullα4 subunitglycine receptorGLRA4hyperekplexiastartle diseasezebrafish |
| spellingShingle | Sophie Leacock Parnayan Syed Victoria M. James Anna Bode Koichi Kawakami Angelo Keramidas Maximiliano Suster Joseph W. Lynch Joseph W. Lynch Robert J. Harvey Robert J. Harvey Structure/Function Studies of the α4 Subunit Reveal Evolutionary Loss of a GlyR Subtype Involved in Startle and Escape Responses Frontiers in Molecular Neuroscience α4 subunit glycine receptor GLRA4 hyperekplexia startle disease zebrafish |
| title | Structure/Function Studies of the α4 Subunit Reveal Evolutionary Loss of a GlyR Subtype Involved in Startle and Escape Responses |
| title_full | Structure/Function Studies of the α4 Subunit Reveal Evolutionary Loss of a GlyR Subtype Involved in Startle and Escape Responses |
| title_fullStr | Structure/Function Studies of the α4 Subunit Reveal Evolutionary Loss of a GlyR Subtype Involved in Startle and Escape Responses |
| title_full_unstemmed | Structure/Function Studies of the α4 Subunit Reveal Evolutionary Loss of a GlyR Subtype Involved in Startle and Escape Responses |
| title_short | Structure/Function Studies of the α4 Subunit Reveal Evolutionary Loss of a GlyR Subtype Involved in Startle and Escape Responses |
| title_sort | structure function studies of the α4 subunit reveal evolutionary loss of a glyr subtype involved in startle and escape responses |
| topic | α4 subunit glycine receptor GLRA4 hyperekplexia startle disease zebrafish |
| url | http://journal.frontiersin.org/article/10.3389/fnmol.2018.00023/full |
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