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

• Published in: The Journal of neuroscience 2003-09, Vol.23 (22), p.8051-8059
• Main Authors: Findlay, Geoffrey S, Phelan, Rachel, Roberts, Michael T, Homanics, Gregg E, Bergeson, Susan E, Lopreato, Gregory F, Mihic, S. John, Blednov, Yuri A, Harris, R. Adron
• Format: Article
• Language: English
• Subjects: Acoustic Stimulation; Amino Acid Substitution; Animals; Behavior, Animal - physiology; Behavioral/Systems/Cognitive; Cells, Cultured; Chlorides - metabolism; Gene Targeting; Glycine Agents - pharmacology; Heterozygote; Homozygote; Humans; Kidney - cytology; Kidney - metabolism; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mice, Transgenic; Molecular Sequence Data; Motor Activity - genetics; Oocytes - metabolism; Patch-Clamp Techniques; Phenotype; Receptors, Glycine - drug effects; Receptors, Glycine - genetics; Reflex, Startle - genetics; Reflex, Startle - physiology; Strychnine - pharmacology; Synaptosomes - metabolism; Xenopus
• ISSN: 0270-6474; 1529-2401
• DOI: 10.1523/jneurosci.23-22-08051.2003

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.