Functional complementation of Glra1(spd-ot), a glycine receptor subunit mutant, by independently expressed C-terminal domains.
The oscillator mouse (Glra1(spd-ot)) carries a 9 bp microdeletion plus a 2 bp microinsertion in the glycine receptor alpha1 subunit gene, resulting in the absence of functional alpha1 polypeptides from the CNS and lethality 3 weeks after birth. Depending on differential use of two splice acceptor sites in exon 9 of the Glra1 gene, the mutant allele encodes either a truncated alpha1 subunit (spd(ot)-trc) or a polypeptide with a C-terminal missense sequence (spd(ot)-elg). During recombinant expression, both splice variants fail to form ion channels. In complementation studies, a tail construct, encoding the deleted C-terminal sequence, was coexpressed with both mutants. Coexpression with spd(ot)-trc produced glycine-gated ion channels. Rescue efficiency was increased by inclusion of the wild-type motif RRKRRH. In cultured spinal cord neurons from oscillator homozygotes, viral infection with recombinant C-terminal tail constructs resulted in appearance of endogenous alpha1 antigen. The functional rescue of alpha1 mutants by the C-terminal tail polypeptides argues for a modular subunit architecture of members of the Cys-loop receptor family.
Villmann
Carmen
C
Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.
Oertel
Jana
J
Ma-Högemeier
Zhan-Lu
ZL
Hollmann
Michael
M
Sprengel
Rolf
R
Becker
Kristina
K
Breitinger
Hans-Georg
HG
Becker
Cord-Michael
CM
D000818:Animals; D019921:Biotinylation / Q000379:methods; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004622:Embryo, Mammalian; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D051379:Mice; D008822:Mice, Transgenic; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation / Q000235:genetics*; D009474:Neurons / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000379:methods; D017434:Protein Structure, Tertiary / Q000235:genetics; D018009:Receptors, Glycine / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D013116:Spinal Cord / Q000166:cytology; D014162:Transfection / Q000379:methods
C000657604:GLRA1 protein, human; C000657605:Glra1 protein, mouse; D008164:Luminescent Proteins; D018009:Receptors, Glycine