Identification Of Hyperekplexia Mutations That Impair The Homopentameric Assembly Of The Glycine Receptor Glyra1

Hyperekplexia or startle disease is a rare monogenic neuromotor disorder characterized by a generalized stiffness in the newborn (“stiff-baby syndrome”) and excessive startle reflexes in response to unexpected acoustic or tactile stimuli. Hyperekplexia-causing mutations have been identified in several genes encoding proteins that collectively participate in glycinergic neurotransmission. The major target of hyperekplexia mutations is the α1 subunit within the glycine receptor family, which consists of five homologous members, four α subunits (GLYRA1-4) and a single β subunit (GLYRB). Glycine receptors are pentameric ligand-gated chloride channels that are predominantly expressed in inhibitory interneurons of the spinal cord and brain stem, where they mediate postsynaptic hyperpolarization. The GLYRA1 can functionally assemble both as a homopentamer and a GLYRA1/GLYRB heteropentamer. Here, we addressed the question whether a loss-of-function of the GLYRA1 may result from a mutation-induced defect in the pentameric assembly. To this end, we individually expressed a total of 42 hyperekplexia-causing GLYRA1 point mutants, each with a C-terminal hexahistidyl tag, in Xenopus laevis oocytes. We labeled the newly synthesized and the plasma membrane-bound GLYRA1 mutants with [35S]methionine and an amine-reactive fluorescent dye, respectively. We purified the recombinant proteins by metal affinity chromatography, and analyzed their oligomeric and monomeric state by blue native polyacrylamide gel electrophoresis (BN-PAGE) and SDS-PAGE, respectively. We also expressed the same constructs for two-electrode voltage clamp recordings. About 40% of the hyperekplexia mutations led to a significant reduction of the homopentameric assembly. The assembly-affecting mutations were predominantly located in the transmembrane helices TM1, TM3 and TM4 that we have previously shown to be crucial for homopentamer formation (Haeger et al., NSMB 17, 90-98, 2010). In contrast, mutations located in the pore-lining TM2 or the loop regions mostly affected glycine-gated channel function but not homopentamerization.