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Review
. 2013 Nov;170(5):933-52.
doi: 10.1111/bph.12335.

Glycine receptor mouse mutants: model systems for human hyperekplexia

Natascha Schaefer  1 , Georg Langlhofer, Christoph J Kluck, Carmen Villmann
Affiliations
Review

Glycine receptor mouse mutants: model systems for human hyperekplexia

Natascha Schaefer et al. Br J Pharmacol. 2013 Nov.

Abstract

Human hyperekplexia is a neuromotor disorder caused by disturbances in inhibitory glycine-mediated neurotransmission. Mutations in genes encoding for glycine receptor subunits or associated proteins, such as GLRA1, GLRB, GPHN and ARHGEF9, have been detected in patients suffering from hyperekplexia. Classical symptoms are exaggerated startle attacks upon unexpected acoustic or tactile stimuli, massive tremor, loss of postural control during startle and apnoea. Usually patients are treated with clonazepam, this helps to dampen the severe symptoms most probably by up-regulating GABAergic responses. However, the mechanism is not completely understood. Similar neuromotor phenotypes have been observed in mouse models that carry glycine receptor mutations. These mouse models serve as excellent tools for analysing the underlying pathomechanisms. Yet, studies in mutant mice looking for postsynaptic compensation of glycinergic dysfunction via an up-regulation in GABAA receptor numbers have failed, as expression levels were similar to those in wild-type mice. However, presynaptic adaptation mechanisms with an unusual switch from mixed GABA/glycinergic to GABAergic presynaptic terminals have been observed. Whether this presynaptic adaptation explains the improvement in symptoms or other compensation mechanisms exist is still under investigation. With the help of spontaneous glycine receptor mouse mutants, knock-in and knock-out studies, it is possible to associate behavioural changes with pharmacological differences in glycinergic inhibition. This review focuses on the structural and functional characteristics of the various mouse models used to elucidate the underlying signal transduction pathways and adaptation processes and describes a novel route that uses gene-therapeutic modulation of mutated receptors to overcome loss of function mutations.

Keywords: domain complementation; glycine receptor; knock-in; mouse models; spontaneous.

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Figures

Figure 1
Figure 1
Homology models of the human (A, blue – α subunit, green – β subunit) and mouse (B, grey – α subunit, yellow – β subunit) Gly receptor subunits based on the crystal structure of the Caenorhabditis elegans glutamate-gated chloride channel (GluCl) solved by Hibbs and Gouaux (2011) showing the mutated amino acid positions affected in hyperekplexia. Two neighbouring subunits of a pentamer are shown. Amino acid exchanges due to recessive mutations, compound heterozygous mutations or deletions with frameshifts resulting in premature stop codons as well as those with no known mode of inheritance are shown as magenta sticks, while amino acid exchanges based on dominant mutations are depicted in orange. (B) Murine knock-in mutations are shown in italics. Note that in the homology models the large intracellular loop domain (ICD) between M helices 3 and 4 was left out as the crystallized GluCl variant also just had a minimal ICD of three amino acids. Therefore, Gly receptor variants having changes in the ICD region are listed, but not represented in the structures modelled.
Figure 2
Figure 2
Dominant hyperekplexia mutants result in changes in pharmacological properties of the Gly receptor α1 channel (modified from Breitinger et al., 2001). (A) The human mutation P250T (Thr) was introduced into the Gly receptor α1 cDNA and transfected into HEK293 cells. Following application of glycine (1 mM), mutant channels desensitize much faster that those of wild-type (wt Pro) controls (non-desensitizing wt compared to 121 ± 6 ms for P250T). (B) EC50 curves for glycine were measured using various glycine concentrations from 5 μM to 10 mM. With P250T, the glycine dose–response curve shows a rightward shift to lower glycine affinities (arrow, reduced by 24-fold compared with wild-type control).
Figure 3
Figure 3
Recessive hyperekplexia mutants have disordered trafficking and protein stability (modified from Villmann et al., 2009b). Comparison of cell surface and intracellular protein of recessive Gly receptors α1 subunit variants from transfected COS7 cells. (A) Quantified reduced surface expression of Gly receptors α1 mutants (14–50% of wt expression) is evident from labelled surface proteins by NHS-SS-biotin. (B) Cell surface expression was less in non-permeabilized transfected HEK293 cells using the monoclonal anti-Gly receptor α1 antibody mAb2b. While M1-mutants (I244N, S231R) are still expressed at the cell surface, only some antigenic clusters were observed for the arginine mutants R252Q and R392H (lower pictures). (C) Protein stability of Gly receptor α1 subunit mutants analysed by pulse-chase radiolabelling. The radiolabelled mutants from transfected HEK293 cells were immunoprecipitated with the monoclonal antibody mAb2b. The intensities of radiolabelled Gly receptor α1 receptor bands from independent experiments (n = 3) were quantified using the Image Quant software (Molecular Dynamics). Wild-type α1 protein and mutants displayed obvious differences in membrane accumulation and half-life of radiolabelled protein. Protein band of 48 kD and intermediate bands (35 kD) are shown. The dashed lines marked by an asterisk (*) indicate the first appearance of the lower MW Gly receptor α1 protein band at 35 kD.
Figure 4
Figure 4
Phenotype of a homozygous oscillator (spdot/spdot) mouse compared with a wild-type control littermate (+/+). Oscillator mice are usually smaller than wild-type controls. Shown are animals at post-natal day (P)19. The oscillator mutatant usually dies by P21. Symptoms start around P15 and develop progressively during P17–P21. Rigidity of the whole body and massive tremor in forelimbs, hindlimbs and even the tail are seen compared with a healthy control mouse.
Figure 5
Figure 5
Domain complementation for rescue of ion channel function (modified from Unterer et al., and Villmann et al., 2009a). (A) Rescue of function experiments of the truncated oscillator Gly receptor α1 variant spdot compared with truncated wild-type α1. When single domains of the Gly receptor α1 were expressed, no functional channels were observed following 3 mM glycine application (upper panel). If the complementation domain (N-myc tail) was co-expressed, functional rescue was observed for the truncated wild type and the oscillator Gly receptor α1 variants (middle and lower panel). (B) Comparison of infected (DIV21; bottom) and uninfected spinal cord neurons isolated from either homozygous wild type (+/+) or oscillator (ot/ot) mice (DIV21 uninfected) with the N-myc tail construct encoded on a pAAV vector. Infected neurons show a signal for the endogenous Gly receptor α1 protein (bottom 3 panels, right picture specific Gly receptor α1 signal in red) that co-localized (merged pictures) with the N-myc tail complementation domain. (C) Functionality of the Gly receptor α1 expressed from three domains. The functionality of the truncated N-terminal domain of the Gly receptor α1 (α1-trc) was not restored by either the M3–M4 loop or the shortened complementation domain (myc-iDΔ62-TM4). Co-expression of all three domains restored Gly receptor function (middle trace).
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