Glycine Receptor Knock-In Mice and Hyperekplexia-Like Phenotypes: Comparisons with the Null Mutant

Abstract

Strychnine-sensitive glycine receptors (GlyRs) inhibit neurotransmission in the spinal cord and brainstem. To better define the function of this receptor in vivo, we constructed a point mutation that impairs receptor function in the alpha1-subunit and compared these knock-in mice to oscillator (spdot) mice lacking functional GlyR alpha1-subunits. Mutation of the serine residue at amino acid 267 to glutamine (alpha1S267Q) results in a GlyR with normal glycine potency but decreased maximal currents, as shown by electrophysiological recordings using Xenopus oocytes. In addition, single-channel recordings using human embryonic kidney 293 cells indicated profoundly altered properties of the mutated GlyR. We produced knock-in mice bearing the GlyR alpha1 S267Q mutation to assess the in vivo consequences of selectively decreasing GlyR efficacy. Chloride uptake into brain synaptoneurosomes from knock-in mice revealed decreased responses to maximally effective glycine concentrations, although wild-type levels of GlyR expression were observed using 3H-strychnine binding and immunoblotting. A profound increase in the acoustic startle response was observed in knock-in mice as well as a "limb clenching" phenotype. In contrast, no changes in coordination or pain perception were observed using the rotarod or hot-plate tests, and there was no change in GABA(A)-receptor-mediated chloride uptake. Homozygous S267Q knock-in mice, like homozygous spdot mice, exhibited seizures and died within 3 weeks of birth. In heterozygous spdot mice, both decreased 3H-strychnine binding and chloride flux were observed; however, neither enhanced acoustic startle responses nor limb clenching were seen. These data demonstrate that a dominant-negative point mutation in GlyR disrupting normal function can produce a more dramatic phenotype than the corresponding recessive null mutation, and provides a new animal model to evaluate GlyR function in vivo.

Occupancy of a Single Anesthetic Binding Pocket Is Sufficient to Enhance Glycine Receptor Function

“…First, currents elicited from oocytes upon application of a maximal concentration of glycine (10 mM) were determined. The magnitudes of these responses provide a measure of the expression level and function of receptors (17). There was a small but significant difference (Ͻ30%) between the maximal currents elicited by homomeric wt and S267C receptors (Fig.…”

“…Culture and transfection of human embryonic kidney 293 cells and single-channel recordings from outside-out patches were conducted as described previously (17). Outside-out macropatch recordings were similar to single-channel recordings except that the patch electrodes had lower resistances (3-6 M⍀) to promote the formation of larger patches.…”

The novel hyperekplexia allele GLRA1(S267N) affects the ethanol site of the glycine receptor

“…In a1S267N receptors, we observed an increased EC 50 for glycine and no change in I max , while a1S267Q channels exhibit a normal EC 50 and reduced I max. 17 The phenotypic presentation in both cases, however, is compatible with dominant hyperekplexia. While ethanol potentiation was virtually absent in HEK293 expressed homomeric a1(S267N) receptors, some ethanol sensitivity is retained in Xenopus oocyte expression, as potentiation depends on the expression system.…”

“…While ethanol potentiation was virtually absent in HEK293 expressed homomeric a1(S267N) receptors, some ethanol sensitivity is retained in Xenopus oocyte expression, as potentiation depends on the expression system. 14,17 This study shows that a disease-related receptor allele harbours the potential to modify drug responses in affected patients by reducing GlyR affinity and efficacy. Given the low phenotypic penetrance of the disease allele GLRA1(S267N) in one of our patients, however, this allelic polymorphism may not be clinically apparent.…”

The action of ethanol on G protein. <i>In silico</i> and cellular/molecular evidences

“…Some studies have reported that mutations can dramatically alter the functional properties of 1GlyR [9]. The discovery that 1GlyR directly interacts with Gβγ has provided secondary supporting evidence for these findings [10,11].…”