Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha 1/gamma 2) and (gamma 2/alpha 1) in combinations with beta 2 and gamma 2

Two gamma -aminobutyric acid, (GABA(A)) receptor chimeras were designed in order to elucidate the structural requirements for GABA, receptor desensiti zation and assembly. The (alpha1/gamma2) and (gamma2/alpha1) chimeric subun its representing the extracellular N-terminal domain of alpha1 or gamma2 an d the remainder of the gamma2 or alpha1 subunits, respectively, were expres sed with beta2 and beta2 gamma2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (alpha1/gamma2)beta2 and (alpha1/ga mma2)beta2 gamma2 but not the (gamma2/alpha1)beta2 and (gamma2/alpha1)beta2 gamma2 subunit combinations formed functional receptor complexes as shown by whole-cell patch-clamp recordings and [H-3]muscimol and [H-3]flunitrazep am binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (alpha1/y2)-containing receptors was pronounced, as opposed to the staining of the (gamma2/alpha1)-containing receptors, which was onl y slightly higher than background. To explain this, the (alpha1/gamma2) and (gamma2/alpha1) chimeras may act like alpha1 and gamma2 subunits, respecti vely, indicating that the extracellular N-terminal segment is important for assembly. However, the (alpha1/beta2) chimeric subunit had characteristics different from the alpha1 subunit, since the (alpha1/gamma2) 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 beta2 or beta2 gamma2. Surprisingly, the (alpha1/gamma2)(g amma2/alpha1)beta2 subunit combination did desensitize, indicating that the C-terminal segment of the alpha1 subunit may be important for desensitizat ion. Moreover, desensitization was observed for the (alpha1/gamma2)beta2 ga mma2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbit al and GABA. (C) 2001 Elsevier Science Ltd. All rights reserved.