GABAA receptor open-state conformation determines non-competitive antagonist binding.

ABSTRACT: The ?-aminobutyric acid (GABA) type A receptor (GABA(A)R) is one of the most important targets for insecticide action. The human recombinant ?3 homomer is the best available model for this binding site and 4-n-[(3)H]propyl-4'-ethynylbicycloorthobenzoate ([(3)H]EBOB) is the preferred non-competitive antagonist (NCA) radioligand. The uniquely high sensitivity of the ?3 homomer relative to the much-less-active but structurally very-similar ?1 homomer provides an ideal comparison to elucidate structural and functional features important for NCA binding. The ?1 and ?3 subunits were compared using chimeragenesis and mutagenesis and various combinations with the ?1 subunit and modulators. Chimera ?3/?1 with the ?3 subunit extracellular domain and the ?1 subunit transmembrane helices retained the high [(3)H]EBOB binding level of the ?3 homomer while chimera ?1/?3 with the ?1 subunit extracellular domain and the ?3 subunit transmembrane helices had low binding activity similar to the ?1 homomer. GABA at 3?M stimulated heteromers ?1?1 and ?1?3 binding levels more than 2-fold by increasing the open probability of the channel. Addition of the ?1 subunit rescued the inactive ?1/?3 chimera close to wildtype ?1?1 activity. EBOB binding was significantly altered by mutations ?1S15'N and ?3N15'S compared with wildtype ?1 and ?3, respectively. However, the binding activity of ?1?1S15'N was insensitive to GABA and ?1?3N15'S was stimulated much less than wildtype ?1?3 by GABA. The inhibitory effect of etomidate on NCA binding was reduced more than 5-fold by the mutation ?3N15'S. Therefore, the NCA binding site is tightly regulated by the open-state conformation that largely determines GABA(A) receptor sensitivity.

SUBMITTER: Chen L 

PROVIDER: S-EPMC3056339 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature