Molecular determinants of desensitization and assembly of the chimeric GABAA receptor subunits (α1/γ2) and (γ2/α1) in combinations with β2 and γ2

Two γ-aminobutyric acid_A (GABA_A) receptor chimeras were designed in order to elucidate the structural requirements for GABA_A receptor desensitization and assembly. The (α1/γ2) and (γ2/α1) chimeric subunits representing the extracellular N-terminal domain of α1 or γ2 and the remainder of the γ2 or α1 subunits, respectively, were expressed with β2 and β2γ2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (α1/γ2)β2 and (α1/γ2)β2γ2 but not the (γ2/α1)β2 and (γ2/α1)β2γ2 subunit combinations formed functional receptor complexes as shown by whole-cell patch–clamp recordings and ³H]muscimol and ³H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (α1/γ2)-containing receptors was pronounced, as opposed to the staining of the (γ2/α1)-containing receptors, which was only slightly higher than background. To explain this, the (α1/γ2) and (γ2/α1) chimeras may act like α1 and γ2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (α1/γ2) chimeric subunit had characteristics different from the α1 subunit, since the (α1/γ2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch–clamp recordings, which was independent of whether the chimera was expressed in combination with β2 or β2γ2. Surprisingly, the (α1/γ2)(γ2/α1)β2 subunit combination did desensitize, indicating that the C-terminal segment of the α1 subunit may be important for desensitization. Moreover, desensitization was observed for the (α1/γ2)β2γ2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.