Compound heterozygosity and nonsense mutations in the alpha(1)-subunit of the inhibitory glycine receptor in hyperekplexia.

The alpha(1)-inhibitory glycine receptor is a ligand-gated chloride channel composed of three ligand-binding alpha1-subunits and two structural beta-subunits that are clustered on the postsynaptic membrane of inhibitory glycinergic neurons. Dominant and recessive mutations in GLRA1 subunits have been associated with a proportion of individuals and families with startle disease or hyperekplexia (MIM: 149400). Following SSCP and bi-directional di-deoxy fingerprinting mutational analysis of 22 unrelated individuals with hyperekplexia and hyperekplexia-related conditions, we report further novel missense mutations and the first nonsense point mutations in GLRA1, the majority of which localise outside the regions previously associated with dominant, disease-segregating mutations. Population studies reveal the unique association of each mutation with disease, and reveals that a proportion of sporadic hyperekplexia is accounted for by the homozygous inheritance of recessive GLRA1 mutations or as part of a compound heterozygote.

A nonsense mutation in the alpha1 subunit of the inhibitory glycine receptor associated with bovine myoclonus.

Inherited congenital myoclonus of Poll Hereford calves is an autosomal recessive disease characterized by hyperesthesia and myoclonic jerks of the skeletal musculature that occur both spontaneously and in response to sensory stimuli. Binding studies have previously shown that myoclonus is associated with specific loss of [(3)H]strychnine-binding sites from spinal cord and brain stem in affected calves. In order to identify the mutation responsible for myoclonus, we examined the candidate genes, glycine receptor alpha1 (Glra1) and beta (Glrb) subunits, in affected and normal cattle. A nonsense mutation was found at amino acid 24, located in exon 2 of the Glra1 gene in both cDNA and genomic sequences from affected but not control animals. Immunohistochemistry, with a monoclonal antibody to alpha and beta subunits of the glycine receptor, revealed a loss of cell surface immunoreactivity in myoclonic animals, suggesting a failure in the assembly of the receptor that could explain the characteristic phenotype of the disease.

Identification of a new ligand binding domain in the alpha1 subunit of the inhibitory glycine receptor.

Four discontinuous extracellular sequence domains have been proposed to form the ligand binding sites of the ligand-gated ion channel receptor superfamily. In this study, we investigated the role of 12 contiguous residues of the inhibitory glycine receptor that define the proposed "loop A" ligand binding domain. Using the techniques of site-directed mutagenesis and patch-clamp electrophysiology, four of the 12 residues were shown to have impaired ligand binding. Three mutants, 193A, A101H, and N102A, resulted in significant (17-44-fold) increases in the agonist EC50 values as compared with the wild-type glycine receptor, whereas Hill coefficients, ImaX values, and antagonist affinity remained largely unaffected. Consideration of receptor efficacy values indicates that these residues are involved in ligand binding rather than channel activation. A fourth mutant, W94A, failed to give rise to any glycine-activated currents, although cell-surface expression was observed, suggesting that this residue may also be involved in agonist binding. These data provide the most extensive characterization of the loop A ligand binding domain available to date and define two new residue locations, Ile93 and Asn102, as contributing to the four-loop model of ligand binding.