REGIONAL GAMMA-AMINOBUTYRIC-ACID SENSITIVITY OF T-BUTYLBICYCLOPHOSPHORO[S-35]THIONATE BINDING DEPENDS ON ALPHA-AMINOBUTYRIC ACID(A) RECEPTOR-ALPHA SUBUNIT

Gamma-Aminobutyric acid (GABA) modulates the convulsant binding site on GABA(A) receptors labeled by t-butylbicyclophosphoro[S-35] thionate ([S-35]TBPS). The modulation varies between different brain regions, reflecting the molecular heterogeneity of the GABA(A) receptors. In rat brain cryostat sections, the main sensitivity difference to GABA between brain regions was observed within the cerebellum. [S-35]TBPS binding in the granule cell layer was more sensitive to GABA than was that in the molecular layer and was detected only after blockade of the GABA agonist sites by the specific GABA(A) antagonists SR 95531, RU 5135, and bicuculline. This indicates that the [S-35]TBPS binding sites in cerebellar granule cells were blocked by endogenous GABA. In contrast, the internal rim of the granule cell layer had a small amount of binding that was largely insensitive to 50 muM GABA. The molecular basis for the sensitivity difference could be traced to the a subunits of the GABA(A) receptor. Expression in human embryonic kidney 293 cells of alpha6beta2gamma2 receptors produced [S-35] TBPS binding sites that were about 10-fold more sensitive to inhibition by GABA than were those inherent to alpha1beta2gamma2 receptors. Coexpression of alpha6 and beta2 subunits produced [S-35]TBPS binding sites that were largely insensitive to GABA inhibition, resembling in their pharmacological profile the sites in the internal granule cell layer. Furthermore, the differences between alpha6beta2 and alpha6beta2gamma2 receptors stress the importance of the gamma2 subunit for the proper pharmacological fingerprint of the rest of the granule cell layer. The neurosteroid 5alpha-pregnan-3alpha-ol-20-one affected the binding in both alpha1beta2gamma2 and alpha6beta2gamma2 receptors, but inhibition was greater in alpha6-containing than in alpha1-containing receptors, suggesting differential coupling of both GABA and neurosteroid sites with the convulsant site. These data might serve as a platform for additional studies to assess the amino acid residues in the two alpha subunits that are critically involved in the allosteric interactions between the GABA(A) agonist/antagonist or neurosteroid domains and the convulsant site.