Glycine receptor knock-in mice and hyperekplexia-like phenotypes: comparisons with the null mutant.
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.
Findlay
Geoffrey S
GS
Waggoner Center for Alcohol and Addiction Research, Section of Neurobiology, University of Texas at Austin, Austin, Texas 78712, USA.
Phelan
Rachel
R
Roberts
Michael T
MT
Homanics
Gregg E
GE
Bergeson
Susan E
SE
Lopreato
Gregory F
GF
Mihic
S John
SJ
Blednov
Yuri A
YA
Harris
R Adron
RA
D000161:Acoustic Stimulation; D019943:Amino Acid Substitution; D000818:Animals; D001522:Behavior, Animal / Q000502:physiology; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D018390:Gene Targeting; D018684:Glycine Agents / Q000494:pharmacology; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009043:Motor Activity / Q000235:genetics; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010641:Phenotype; D018009:Receptors, Glycine / Q000187:drug effects / Q000235:genetics*; D013216:Reflex, Startle / Q000235:genetics / Q000502:physiology; D013331:Strychnine / Q000494:pharmacology; D013574:Synaptosomes / Q000378:metabolism; D014981:Xenopus
D002712:Chlorides; D018684:Glycine Agents; D018009:Receptors, Glycine; D013331:Strychnine