Abstract
Startle disease is a rare, potentially fatal neuromotor disorder characterized by exaggerated startle reflexes and hypertonia in response to sudden unexpected auditory, visual or tactile stimuli. Mutations in the GlyR α1 subunit gene (GLRA1) are the major cause of this disorder, since remarkably few individuals with mutations in the GlyR β subunit gene (GLRB) have been found to date. Systematic DNA sequencing of GLRB in individuals with hyperekplexia revealed new missense mutations in GLRB, resulting in M177R, L285R and W310C substitutions. The recessive mutation M177R results in the insertion of a positively-charged residue into a hydrophobic pocket in the extracellular domain, resulting in an increased EC50 and decreased maximal responses of α1β GlyRs. The de novo mutation L285R results in the insertion of a positively-charged side chain into the pore-lining 9′ position. Mutations at this site are known to destabilize the channel closed state and produce spontaneously active channels. Consistent with this, we identified a leak conductance associated with spontaneous GlyR activity in cells expressing α1βL285R GlyRs. Peak currents were also reduced for α1βL285R GlyRs although glycine sensitivity was normal. W310C was predicted to interfere with hydrophobic side-chain stacking between M1, M2 and M3. We found that W310C had no effect on glycine sensitivity, but reduced maximal currents in α1β GlyRs in both homozygous (α1βW310C) and heterozygous (α1ββW310C) stoichiometries. Since mild startle symptoms were reported in W310C carriers, this may represent an example of incomplete dominance in startle disease, providing a potential genetic explanation for the ‘minor’ form of hyperekplexia.
Highlights
Inhibitory glycine receptors (GlyRs) are ligand-gated chloride channels enriched in the spinal cord, brainstem and retina, consisting of heteropentameric combinations of ligand-binding GlyR α subunits (α1–α4) together with the GlyR β subunit (Lynch, 2009)
Individuals with startle disease lacking mutations in GLRA1 and SLC6A5 were screened for genetic variation in GLRB coding exons and donor and acceptor splice junctions by Sanger DNA sequencing
We found that the reduction of Cl- flux was dependent on the ratio of wild-type GlyR α1 subunit to mutant GlyR β subunits (Fig. 7D), with significantly reduced Cl- flux observed with increasing levels of βW310C expression
Summary
Inhibitory glycine receptors (GlyRs) are ligand-gated chloride channels enriched in the spinal cord, brainstem and retina, consisting of heteropentameric combinations of ligand-binding GlyR α subunits (α1–α4) together with the GlyR β subunit (Lynch, 2009). Experiments with GlyR α1-β subunit concatemers revealed that functional heteromeric GlyRs can be produced when these were co-expressed with GlyR β subunit monomers but not when expressed alone, or with GlyR α1 subunit monomers (Grudzinska et al, 2005). This result was consistent with either a 2α:3β or a 1α:4β stoichiometry. Quantification of (35S)methionine incorporation into recombinant α1β versus α1 subunit GlyRs suggested a 2α:3β stoichiometry and the subunit order β-α-β-α-β This stoichiometry and subunit arrangement has recently been confirmed by imaging single antibody-bound GlyR α1β heteromers using atomic force microscopy (Yang et al, 2012)
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