Abstract
Key points Hyperekplexia or startle disease is a serious neurological condition affecting newborn children and usually involves dysfunctional glycinergic neurotransmission.Glycine receptors (GlyRs) are major mediators of inhibition in the spinal cord and brainstem.A missense mutation, replacing asparagine (N) with lysine (K), at position 46 in the GlyR α1 subunit induced hyperekplexia following a reduction in the potency of the transmitter glycine; this resulted from a rapid deactivation of the agonist current at mutant GlyRs.These effects of N46K were rescued by mutating a juxtaposed residue, N61 on binding Loop D, suggesting these two asparagines may interact.Asparagine 46 is considered to be important for the structural stability of the subunit interface and glycine binding site, and its mutation represents a new mechanism by which GlyR dysfunction induces startle disease. Dysfunctional glycinergic inhibitory transmission underlies the debilitating neurological condition, hyperekplexia, which is characterised by exaggerated startle reflexes, muscle hypertonia and apnoea. Here we investigated the N46K missense mutation in the GlyR α1 subunit gene found in the ethylnitrosourea (ENU) murine mutant, Nmf11, which causes reduced body size, evoked tremor, seizures, muscle stiffness, and morbidity by postnatal day 21. Introducing the N46K mutation into recombinant GlyR α1 homomeric receptors, expressed in HEK cells, reduced the potencies of glycine, β‐alanine and taurine by 9‐, 6‐ and 3‐fold respectively, and that of the competitive antagonist strychnine by 15‐fold. Replacing N46 with hydrophobic, charged or polar residues revealed that the amide moiety of asparagine was crucial for GlyR activation. Co‐mutating N61, located on a neighbouring β loop to N46, rescued the wild‐type phenotype depending on the amino acid charge. Single‐channel recording identified that burst length for the N46K mutant was reduced and fast agonist application revealed faster glycine deactivation times for the N46K mutant compared with the WT receptor. Overall, these data are consistent with N46 ensuring correct alignment of the α1 subunit interface by interaction with juxtaposed residues to preserve the structural integrity of the glycine binding site. This represents a new mechanism by which GlyR dysfunction induces startle disease.
Highlights
Hyperekplexia or startle disease is a serious neurological condition affecting newborn children
The underlying cause of hyperekplexia involves dysfunctional glycinergic transmission (Harvey et al 2008) and causative mutations are typically found in the genes encoding glycine receptor (GlyR) α1 (GLRA1; Shiang et al 1993, 1995; Chung et al 2010) and β subunits (GLRB; Rees et al 2002), and the presynaptic glycine transporter, GlyT2 (Rees et al 2006)
Animal models of startle disease are crucial for understanding the complex genetics of hyperekplexia and characteristic symptoms are exhibited by several mouse mutants harbouring different mutations in the GlyR α1 subunit gene (GLRA1), including: spasmodic, oscillator, cincinatti (Graham et al 2006; Schaefer et al 2013) and Nmf11 (Traka et al 2006)
Summary
Hyperekplexia or startle disease is a serious neurological condition affecting newborn children It is characterised by exaggerated startle reflexes following tactile and acoustic stimuli, resulting in hypertonia and apnoea. Animal models of startle disease are crucial for understanding the complex genetics of hyperekplexia and characteristic symptoms are exhibited by several mouse mutants harbouring different mutations in the GlyR α1 subunit gene (GLRA1), including: spasmodic, oscillator, cincinatti (Graham et al 2006; Schaefer et al 2013) and Nmf (Traka et al 2006). A missense mutation (A52S) in the extracellular domain of GlyR α1, caused a relatively mild phenotype, with homozygous mice appearing normal at rest but developing an exaggerated startle response to acoustic or tactile stimuli at around 2 weeks of age (Lane et al 1987). Our data identify an approximate threshold for the reduction in glycine potency that results in lethality of GlyR mutant mice, in addition to uncovering a role for N46 in GlyR activation/ deactivation and a new mechanism for hyperekplexia
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