Research Article: Individual knock out of glycine receptor alpha subunits identifies a specific requirement of glra1 for motor function in zebrafish

Date Published: May 2, 2019

Publisher: Public Library of Science

Author(s): Eric Samarut, Domitille Chalopin, Raphaëlle Riché, Marc Allard, Meijiang Liao, Pierre Drapeau, Michael Klymkowsky.


Glycine receptors (GlyRs) are ligand-gated chloride channels mediating inhibitory neurotransmission in the brain stem and spinal cord. They function as pentamers composed of alpha and beta subunits for which 5 genes have been identified in human (GLRA1, GLRA2, GLRA3, GLRA4, GLRB). Several in vitro studies showed that the pentameric subtype composition as well as its stoichiometry influence the distribution and the molecular function of the receptor. Moreover, mutations in some of these genes are involved in different human conditions ranging from tinnitus to epilepsy and hyperekplexia, suggesting distinct functions of the different subunits. Although the beta subunit is essential for synaptic clustering of the receptor, the specific role of each alpha subtype is still puzzling in vivo. The zebrafish genome encodes for five glycine receptor alpha subunits (glra1, glra2, glra3, glra4a, glra4b) thus offering a model of choice to investigate the respective role of each subtype on general motor behaviour. After establishing a phylogeny of GlyR subunit evolution between human and zebrafish, we checked the temporal expression pattern of these transcripts during embryo development. Interestingly, we found that glra1 is the only maternally transmitted alpha subunit. We also showed that the expression of the different GlyR subunits starts at different time points during development. Lastly, in order to decipher the role of each alpha subunit on the general motor behaviour of the fish, we knocked out individually each alpha subunit by CRISPR/Cas9-targeted mutagenesis. Surprisingly, we found that knocking out any of the alpha2, 3, a4a or a4b subunit did not lead to any obvious developmental or motor phenotype. However, glra1-/- (hitch) embryos depicted a strong motor dysfunction from 3 days, making them incapable to swim and thus leading to their premature death. Our results infer a strong functional redundancy between alpha subunits and confirm the central role played by glra1 for proper inhibitory neurotransmission controlling locomotion. The genetic tools we developed here will be of general interest for further studies aiming at dissecting the role of GlyRs in glycinergic transmission in vivo and the hitch mutant (hic) is of specific relevance as a new model of hyperekplexia.

Partial Text

Glycine receptors (GlyRs) are ligand-activated chloride channels mediating fast inhibitory neurotransmission in the adult brain stem and spinal cord. GlyRs depict a modular structure with a N-terminal extracellular glycine binding domain followed by four transmembrane domains (M1 to M4) at their C-terminal part and a long intracellular loop between M3 and M4. GlyRs function as pentamers composed of different beta and/or alpha subunits that form a pore letting chloride ions flow through the cell membrane once opened. Five GlyR subunit genes have been referenced in mammals consisting of 4 alpha (GLRA1, GLRA2, GLRA3, GLRA4) and one beta subunit (GLRB) and these genes can be considered as paralogs. Of note is that GLRA4 is considered as a pseudogene in human. Interestingly, chloride conductance and other channel molecular features are influenced by the subunit composition as well as their very diverse stoichiometry (3α1:2β, 2α1:3β, or 1α1:4β) [1–4]. Although heteromeric channels are thought to mediate most of the glycinergic neurotransmission in the adult [5–8], GlyRs can also form homomeric pentamers of alpha subunits only. These homomeric channels are believed to be extra-synaptic [9, 10] since only the beta subunit is able to bind to gephyrin, an anchoring protein necessary for GlyR localization at the synapse [11, 12].

In this work, we aimed at investigating the specific function during locomotion of each glycine receptor alpha subunits in vivo, taking advantage of the zebrafish embryo. Unexpectedly, we noticed a strong functional redundancy between glra2, a3, a4a and a4b different subunits, with alpha 1 being the only one specifically required for correct glycine inhibitory neurotransmission involved in motor function. This is consistent with data obtained from mice models lacking the alpha 1 subunit that depict a motor dysfunction reminiscent to hyperekplexia [13]. The fact that knocking-out the other alpha subunits did not lead to a major motor phenotype is also consistent with the fact that glra2-KO and glra3-KO mice do not display motility defects but rather inflammatory and sensory dysfunctions [14, 15]. At this juncture, our new genetic lines bearing homozygous knockout mutations in each individual a2, a3, a4a and a4b subunit could be of interest for further studying the role of glycinergic transmission in a broad variety of physiological processes, especially during development due to the ease of studying the zebrafish embryo. Moreover, although beyond the scope of the present study, these lines could be further used to characterize the electrophysiological involvement of each alpha subunit on the channel activity. This would allow to further describe the specific function of each subunit at the molecular level. Moreover, we believe that the hitch mutant lacking the expression of the alpha 1 subunit is an interesting model to complement the genetic tools already available to investigate hyperekplexia and other glycine-related disorders. Indeed, one third of the patients with hyperekplexia carry a loss-of-function mutation in the alpha 1 subunit (GLRA1) of the glycine receptor and such a genetic condition has not been previously modeled in zebrafish. Thus, our hitch (hic) mutant complements the library of zebrafish genetic models with glycinergic transmission defects that are bandoneon mutants (beo) knocked-out for glrbb [23] and shocked mutants (sho) knocked-out for glycine transporter 1 (glyT1) [34].




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