Pathophysiological Mechanisms of Dominant and Recessive GLRA1 Mutations in Hyperekplexia

Abstract

Hyperekplexia is a rare, but potentially fatal, neuromotor disorder characterized by exaggerated startle reflexes and hypertonia in response to sudden, unexpected auditory or tactile stimuli. This disorder is primarily caused by inherited mutations in the genes encoding the glycine receptor (GlyR) ␣1 subunit (GLRA1) and the presynaptic glycine transporter GlyT2 (SLC6A5). In this study, systematic DNA sequencing of GLRA1 in 88 new unrelated human hyperekplexia patients revealed 19 sequence variants in 30 index cases, of which 21 cases were inherited in recessive or compound heterozygote modes. This indicates that recessive hyperekplexia is far more prevalent than previous estimates. From the 19 GLRA1 sequence variants, we have investigated the functional effects of 11 novel and 2 recurrent mutations. The expression levels and functional properties of these hyperekplexia mutants were analyzed using a high-content imaging system and patch-clamp electrophysiology. When expressed in HEK293 cells, either as homomeric ␣1 or heteromeric ␣1␤ GlyRs, subcellular localization defects were the major mechanism underlying recessive mutations. However, mutants without trafficking defects typically showed alterations in the glycine sensitivity suggestive of disrupted receptor function. This study also reports the first hyperekplexia mutation associated with a GlyR leak conductance, suggesting tonic channel opening as a new mechanism in neuronal ligand-gated ion channels.

New Hyperekplexia Mutations Provide Insight into Glycine Receptor Assembly, Trafficking, and Activation Mechanisms

“…All sequence variations were cross-referenced with the dbSNP database and our previous GLRA1 data sets (for recurrent mutations) and were regarded as probable mutations following exclusion from a panel of 100 control samples and the exome variants server. The screening revealed 19 mutations in 21 hyperekplexia probands (Table 1), a rate that is consistent with previous studies (10,28). Nine mutations were novel in the public domain, and three (one novel and two recurrent) mutations were present in more than one individual.…”

“…Additionally, confirmation of the recurrent exon 1-7 deletion mutation was carried out using break point PCR analysis (19). Mutations were introduced into pRK5-hGlyR␣1 using the QuikChange site-directed mutagenesis kit (Stratagene) and confirmed by direct sequencing of the entire transgene-coding region (10).…”

“…We previously presented the results of a sequencing screen of the GlyR ␣1 subunit (GLRA1) in 88 unrelated hyperekplexia probands from where we identified a total of 19 mutations, 12 of which were novel (10). We demonstrated that dominant mutations typically disrupt receptor function without changing their surface expression efficiency, whereas recessive mutations generally preclude functional receptor expression when expressed either as mutated ␣1 homomers or as heteromers with the ␤ subunit.…”

“…All new mutations were characterized in terms of their electrophysiological properties and their cell surface localization. Additionally, a previously characterized mutation, p.R65W (10), was reinvestigated because of its presumed compound heterozygosity with the new mutation, p.P230S. The subsequent functional investigation of the hyperekplexia mutations revealed three residues critical for GlyR channel opening, providing insight into pLGIC activation mechanisms, and one novel residue playing an important role in desensitization.…”

Stoichiometry and Subunit Arrangement of α1β Glycine Receptors As Determined by Atomic Force Microscopy

“…As they are considered potential therapeutic targets for indications including inflammatory pain, hyperekplexia, spasticity, tinnitus and breathing disorders [2][3][4][5] , there is widespread interest in understanding the molecular structure of their ligand binding sites for therapeutic development. GlyRs are members of the pentameric Cys-loop ion channel receptor family.…”