The channel-lining 6' amino acid in the second membrane-spanning region of ionotropic GABA receptors has more profound effects on 4'-ethynyl-4-n-propylbicycloorthobenzoate binding than the 2' amino acid.
The noncompetitive antagonist of ionotropic gamma-aminobutyric acid (GABA) receptors 4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) is a useful tool to probe the antagonist-binding site. In the present study, four mutants of the human GABA(A) receptor beta3 subunit were stably expressed in S2 cells and examined for their abilities to bind [(3)H]EBOB to identify the binding site of EBOB. The homo-oligomeric beta3 GABA receptor was used as a housefly GABA receptor model, as the beta3 subunit has a high sequence similarity with the housefly Rdl subunit in the second membrane-spanning (M2) region. The A274S mutation at the -1' position in the M2 region had no effect on [(3)H]EBOB binding. The A277S mutation at the 2' position led to a decrease in the affinity of EBOB for the GABA receptor. The T281V mutant at the 6' position and the A277S/T281V double mutant completely abolished the binding ability. A beta3 GABA receptor homology model predicts these interactions between the receptor and EBOB. These results suggest that EBOB interacts with threonine 281 and alanine 277, and that threonine 281 plays a more critical role in interacting with EBOB than alanine 277.
Hisano
Kazutoshi
K
Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane, 690-8504, Japan.
Ozoe
Fumiyo
F
Huang
Jia
J
Kong
Xiangyu
X
Ozoe
Yoshihisa
Y
D019943:Amino Acid Substitution; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000737:chemistry / Q000494:pharmacology*; D017931:DNA Primers; D006801:Humans; D007306:Insecticides / Q000494:pharmacology*; D008958:Models, Molecular; D008968:Molecular Conformation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011963:Receptors, GABA-A / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D013329:Structure-Activity Relationship; D014316:Tritium
D019086:Bridged Bicyclo Compounds, Heterocyclic; D017931:DNA Primers; D007306:Insecticides; D011963:Receptors, GABA-A; D014316:Tritium; C091518:1-(4-ethynylphenyl)-4-propyl-2,6,7-trioxabicyclo(2.2.2)octane