The F-loop of the GABA A receptor gamma2 subunit contributes to benzodiazepine modulation.
GABA(A) receptors can be modulated by benzodiazepines, although these compounds do not directly activate or inhibit the receptors. The prototypic benzodiazepine, diazepam, potentiates responses to GABA in GABA(A) receptors that contain a gamma subunit. Here we have used mutagenesis, radioligand binding, voltage clamp electrophysiology, and homology modeling to probe the role of the F-loop residues Asp(192)-Arg(197) in the GABA(A) receptor gamma(2) subunit in diazepam potentiation of the GABA response. Substitution of all of these residues with Ala and/or a residue with similar chemical properties to the wild type residue decreased the level of diazepam potentiation, and one mutation (D192A) resulted in its complete ablation. None of the mutations changed the GABA EC(50) or the [(3)H]flumazenil binding affinity, suggesting they do not affect GABA or benzodiazepine binding characteristics; we therefore propose that they are involved in the diazepam-mediated conformational change that results in an increased response to GABA. Homology models of the receptor binding pocket in agonist-bound and unbound states suggest that the F-loop is flexible and has different orientations in the two states. Considering our data in relation to these models, we find that the F-loop residues could contribute to hydrogen bond networks and hydrophobic interactions with neighboring residues that change during receptor activation.
Padgett
Claire L
CL
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, United Kingdom.
Lummis
Sarah C R
SC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D003975:Diazepam / Q000494:pharmacology*; D005260:Female; D018757:GABA Modulators / Q000494:pharmacology*; D006801:Humans; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D011869:Radioligand Assay; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D018757:GABA Modulators; C546279:GABRG2 protein, human; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D003975:Diazepam