Repression of gamma -aminobutyric acid type A receptor alpha 1 polypeptide biosynthesis requires chronic agonist exposure

Although it is well established that the number of gamma -aminobutyric acid type A (GABA sub(A)) receptors declines in cortical neurons exposed to GABA sub(A) receptor agonists, the mechanisms responsible for this use-dependent down-regulation remain unclear. Two hypotheses have been proposed: (i) agonist-evoked sequestration and degradation of surface GABA sub(A) receptors and (ii) repression of receptor subunit biosynthesis. We have addressed this problem using [ super(35)S]Met/Cys pulse-chase labeling of chick cortical neurons in culture and immunoprecipitation and immunoblotting with an antibody (RP4) directed against a GABA sub(A) receptor alpha 1-(331-381) fusion protein. Exposure of the cells to GABA or isoguvacine for 2 h to 4 days had no effect on the initial rate of super(35)S incorporation into the GABA sub(A) receptor 51-kDa alpha 1 polypeptide, but this rate declined by 33% after a 7-day treatment. This is consistent with a previous report that a 7-day GABA treatment of this preparation produced a 45% reduction in the alpha 1 subunit mRNA level, while a 4-day exposure had no detectable effect. On the other hand, after a 4-day exposure to these agonists, a 30% reduction in the level of the alpha 1 polypeptide was observed on immunoblots, similar to that found previously for down-regulation of GABA sub(A) receptor ligand-binding sites. Thus, the de novo synthesis of GABA sub(A) receptor alpha 1 subunits is subject to a delayed use-dependent repression that was observed after, rather than before, the decline in neuronal levels of the polypeptide. Pulse-chase experiments showed a monophasic degradation of the GABA sub(A) receptor super(35)S- alpha 1 subunit with a t sub( one half ) = 7.7 h, a process that was unaffected by the addition of GABA to neurons during the chase period. These nascent super(35)S-labeled polypeptides are presumably diluted into the neuronal pool of unlabeled unassembled alpha 1 subunits, which was found to exceed by a 4:1 molar ratio the amount assembled into [ super(3)H]flunitrazepam binding sites. Thus, the data reveal an alternative scheme for degradation of GABA sub(A) receptor polypeptides: a pathway that may participate in the agonist-independent degradation of unassembled receptor subunits. This differs from another pathway for the agonist-dependent degradation of mature GABA sub(A) receptors derived from the neuronal surface.