TY - JOUR
T1 - Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha1/gamma2) and (gamma2/alpha1) in combinations with beta2 and gamma2
AU - Elster, L
AU - Kristiansen, U
AU - Pickering, D S
AU - Olsen, R W
AU - Schousboe, A
N1 - Keywords: Animals; Cell Line; Rats; Receptors, GABA-A; Recombinant Fusion Proteins; Spodoptera
PY - 2001
Y1 - 2001
N2 - Two gamma-aminobutyric acid(A) (GABA(A)) receptor chimeras were designed in order to elucidate the structural requirements for GABA(A) receptor desensitization and assembly. The (alpha1/gamma2) and (gamma2/alpha1) chimeric subunits representing the extracellular N-terminal domain of alpha1 or gamma2 and the remainder of the gamma2 or alpha1 subunits, respectively, were expressed with beta2 and beta2gamma2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (alpha1/gamma2)beta2 and (alpha1/gamma2)beta2gamma2 but not the (gamma2/alpha1)beta2 and (gamma2/alpha1)beta2gamma2 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 (alpha1/gamma2)-containing receptors was pronounced, as opposed to the staining of the (gamma2/alpha1)-containing receptors, which was only slightly higher than background. To explain this, the (alpha1/gamma2) and (gamma2/alpha1) chimeras may act like alpha1 and gamma2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (alpha1/gamma2) 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 beta2gamma2. Surprisingly, the (alpha1/gamma2)(gamma2/alpha1)beta2 subunit combination did desensitize, indicating that the C-terminal segment of the alpha1 subunit may be important for desensitization. Moreover, desensitization was observed for the (alpha1/gamma2)beta2gamma2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.
AB - Two gamma-aminobutyric acid(A) (GABA(A)) receptor chimeras were designed in order to elucidate the structural requirements for GABA(A) receptor desensitization and assembly. The (alpha1/gamma2) and (gamma2/alpha1) chimeric subunits representing the extracellular N-terminal domain of alpha1 or gamma2 and the remainder of the gamma2 or alpha1 subunits, respectively, were expressed with beta2 and beta2gamma2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (alpha1/gamma2)beta2 and (alpha1/gamma2)beta2gamma2 but not the (gamma2/alpha1)beta2 and (gamma2/alpha1)beta2gamma2 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 (alpha1/gamma2)-containing receptors was pronounced, as opposed to the staining of the (gamma2/alpha1)-containing receptors, which was only slightly higher than background. To explain this, the (alpha1/gamma2) and (gamma2/alpha1) chimeras may act like alpha1 and gamma2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (alpha1/gamma2) 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 beta2gamma2. Surprisingly, the (alpha1/gamma2)(gamma2/alpha1)beta2 subunit combination did desensitize, indicating that the C-terminal segment of the alpha1 subunit may be important for desensitization. Moreover, desensitization was observed for the (alpha1/gamma2)beta2gamma2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.
M3 - Journal article
C2 - 11290383
SN - 0197-0186
VL - 38
SP - 581
EP - 592
JO - Neurochemistry International
JF - Neurochemistry International
IS - 7
ER -